High efficiency, small volume nucleic acid synthesis

ABSTRACT

The disclosure generally relates to compositions and methods for the production of nucleic acid molecules. In some aspects, the invention allows for the microscale generation of nucleic acid molecules, optionally followed by assembly of these nucleic acid molecules into larger molecules. In some aspects, the invention allows for efficient production of nucleic acid molecules (e.g., large nucleic acid molecules such as genomes).

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No. 15/463,363filed on Mar. 20, 2017, now allowed, which is a divisional of U.S.application Ser. No. 14/730,824 filed on Jun. 4, 2015, now abandoned,which is a continuation of U.S. application Ser. No. 13/627,819 filedSep. 26, 2012, now abandoned, which claims priority to U.S. ApplicationNo. 61/539,303 filed Sep. 26, 2011, which disclosures are hereinincorporated by reference in their entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Aug. 18, 2015, isnamed LT00586CON_SL.txt and is 4,183 bytes in size.

FIELD OF THE INVENTION

The disclosure generally relates to compositions and methods for theproduction of nucleic acid molecules. In some aspects, the inventionallows for the microscale generation of nucleic acid molecules,optionally followed by assembly of these nucleic acid molecules intolarger molecules. In some aspects, the invention allows for efficientproduction of nucleic acid molecules (e.g., large nucleic acid moleculessuch as genomes).

BACKGROUND

Production of nucleic acid molecules can be fairly simple or complexdepending on factors such as the type of nucleic acid molecules to beproduced. For example, historically, short single stranded nucleic acidmolecules such as primers have been typically generated by chemicalsynthesis (see, e.g., U.S. Pat. No. 5,837,858, the disclosure of whichis incorporated herein by reference). Further, longer nucleic acidmolecules have typically been generated by polymerase chain reaction(PCR). One disadvantage of PCR is that generally template nucleic acidis required.

Many nucleic acid synthesis methods have limited capabilities for thegeneration of large de novo nucleic acid molecules. One aspect of thecurrent disclosure is to address this limitation.

SUMMARY OF THE INVENTION

The invention relates, in part, to compositions and methods for thesynthesis of nucleic acid molecules. The invention further relates tocompositions and methods for the assembly of nucleic acid molecules toform molecules such as plasmids, chromosomes and genomes.

In some aspects, the invention relates to multiwell plates fornon-template directed synthesis of nucleic acid molecules. In someembodiments, the plate comprises a bead (e.g., a magnetic bead) locatedin each of a plurality of wells of the plate and an electrochemicallygenerated acid (EGA) being present in one or more of the plurality ofwells. Instead of or in addition to having EGA in one or more wells,wells of the plate may contain other reagents set out elsewhere whereinassociated with the synthesis of nucleic acid molecules.

Bead sizes used in the practice of the invention may vary widely butinclude beads with diameters between 0.01 μm and 100 μm, 0.005 μm and100 μm, 0.005 μm and 10 μm, 0.01 μm and 100 μm, 0.01 μm and 1,000 μm,between 1.0 μm and 2.0 μm, between 1.0 μm and 100 μm, between 2.0 μm and100 μm, between 3.0 μm and 100 μm, between 0.5 μm and 50 μm, between 0.5μm and 20 μm, between 1.0 μm and 10 μm, between 1.0 μm and 20 μm,between 1.0 μm and 30 μm, between 10 μm and 40 μm, between 10 μm and 60μm, between 10 μm and 80 μm, or between 0.5 μm and 10 μm. As one skilledin the art would recognize, when solid particle fall below a particularsize, they begin to act acquire attributes of fluids (e.g., form theequivalent of colloidal suspensions). Thus, in some instances (e.g.,with the use of beads below about 500 nm in diameter), it may bedesirable to treat the bead as a fluid. This may mean removal of a beadfrom a magnetic tip, for example, by agitation, washing, or with the useof a surfactant.

In specific embodiments of the invention, the bead size may be chosendepending on the size of the well to allow only one single bead tooccupy a well. In other embodiments, more than one bead (or nucleic acidsynthesis substrates of other shapes) may be in some of all of thewells. In some instances, the number beads per well may be between twoand twenty, between two and thirty, between two and ten, between fourand twenty, between four and ten, between four and fifty, etc.

The number of wells may also vary widely and is limited by factors suchas the amount of nucleic acid to be produced and technical factors suchas manufacturability and mechanic factors related to use (e.g., thelower size limit of magnetic bead extractors). In any event, the numberof wells may be in number, for example, between 10 and 10,000,000,between 10 and 5,000,000, between 10 and 2,000,000, between 10 and1,000,000, between 10 and 800,000, between 10 and 650,000, between 10and 500,000, between 500 and 500,000, between 10 and 50,000, between1,000 and 500,000, between 10,000 and 500,000, between 20,000 and500,000, or between 1,000 and 50,000. Further, multiwell surfaces havebeen prepared with wells numbering in the range of 10 million. Thus,under some instances, the number of wells may be less than 5 million, 10million, 20 million, etc.

The total volume of each well is another item which may vary and may be,for example, between 1.0×10⁻⁹ μl and 50 μl, between 1.0×10⁻⁹ μl and 10μl, between 1.0×10⁻⁹ μl and 1.0 μl, between 1.0×10⁻⁹ μl and 0.1 μl,between 1.0×10⁻⁹ μl and 1.0×10⁻² μl, between 1.0×10⁻⁹ μl and 1.0×10⁻³μl, between 1.0×10⁻⁹ μl and 1.0×10⁻⁴ μl, between 1.0×10⁻⁹ μl and 50 μl,between 1.0×10⁻⁵ μl and 1.0×10⁻⁶ μl, between 1.0×10⁻⁹ μl and 1.0×10⁻⁷μl, between 2.5×10⁻⁹ μl and 1.0×10⁻² μl, between 2.5×10⁻⁹ μl and1.0×10⁻³ μl, between 2.5×10⁻⁹ μl and 1.0×10⁻⁴ μl, between 2.5×10⁻⁹ μland 1.0×10⁻⁵ μl, between 2.5×10⁻⁴ μl and 1.0×10⁻⁶ μl, between 1.0×10⁻⁸μl and 1.0×10⁻⁶ μl, between 1.0×10⁻⁸ μl and 1.0×10⁻⁵ μl, between1.0×10⁻⁷ μl and 1.0×10⁻⁵ μl, between 1.0×10⁻⁷ μl and 1.0×10⁻⁴ μl,between 1.0×10⁻⁷ μl and 1.0×10⁻³ μl, between 1.0×10⁻⁷ μl and 1.0×10⁻²μl, between 0.1 μl and 50 μl, between 0.01 μl and 50 μl, between 0.01 μland 25 μl, between 0.01 μl and 15 μl, between 0.01 μl and 10 μl, between0.001 μl and 50 μl, between 0.001 μl and 5 μl, between 0.001 μl and 1μl, between 0.001 μl and 0.01 μl, or between 0.001 μl and 1 μl.

In many instances, multiwell plates of the invention or multiwell platessuitable for use with the invention will be operably connected to eitherone electrode or a set (e.g., one or several pairs) of electrodes. Asdiscussed elsewhere herein, these electrodes can be used to generate amicroenvironment associated with catalysis of one or more chemicalreactions (e.g., EGA for nucleotide deprotection).

In some embodiments, multiwell plates of the invention or multiwellplates suitable for use with the invention will be connected tomicrofluidic channels for the introduction and removal of reagents. Thisallows for efficient and automated controlling of reagents.

The invention also provides method for the generation of assemblednucleic acid molecules formed from smaller chemically synthesizednucleic acid molecules. In some embodiments, such method may compriseone or more of the following steps:

(a) synthesizing a plurality of nucleic acid molecules, wherein eachnucleic acid molecule is prepared in a microquantity in the well of aplate;

(b) combining the nucleic acid molecules generated in (a), or a portionthereof, to produce a pool;

(c) joining some or all of the nucleic acid molecules present in thepool formed in (b) to form a plurality of larger nucleic acid molecules;

(d) eliminating nucleic acid molecules which contain sequence errorsfrom the plurality of larger nucleic acid molecules formed in (c) toproduce an error corrected nucleic acid molecule pool; and

(e) assembling the nucleic acid molecules in the error corrected nucleicacid molecule pool to form the assembled nucleic acid molecule.

In some embodiments, the joining of nucleic acid molecules present inthe pool will be mediated by polymerase chain reaction (PCR).

In some embodiments step (b) may further comprise combining nucleic acidmolecules generated in (a) with nucleic acid molecules obtained by othermeans to form a pool, wherein said other means include PCR, restrictionenzyme digest or exonuclease treatment. In some instances, the assemblednucleic acid molecule generated in (c) and/or (e) may be assembled andintroduced into a vector (e.g., a cloning vector, a destination vector,etc.).

The number of nucleic acid molecules assembled by methods of theinvention can vary and, when appropriate, will correlate with the numberof pooled nucleic acid molecules. In any event, nucleic acid moleculesassembled in methods of the invention may be composed of at least fiveother (e.g., smaller) nucleic acid molecules (e.g., from about five toabout five thousand, from about five to about twenty thousand, fromabout five to about one hundred thousand, from about fifty to about fivethousand, from about fifty to about twenty thousand, from about fifty toabout one hundred thousand, from about one hundred to about fivethousand, from about one hundred to about one hundred thousand, fromabout five hundred to about five thousand, from about five hundred toabout one hundred thousand, etc. nucleic acid molecules).

Nucleic acid molecules assembled by methods of the invention may varygreatly and include molecules of at least 20 kilobases (e.g., betweenfrom about 0.5 kilobase and to about 10 megabases, between from about0.5 kilobase and to about 5 megabases, between from about 0.5 kilobaseand to about 1 megabase, between from about 0.5 kilobase and to about500 kilobases, between from about 0.5 kilobase and to about 100kilobases, between from about 0.5 kilobase and to about 10 megabases,between from about 0.5 kilobase and to about 1 kilobase, between fromabout 1 kilobase and to about 10 megabases, between from about 10kilobases and to about 5 megabases, between from about 1 kilobase and toabout 5 megabases, between from about 1 kilobase and to about 2megabases, between from about 1 kilobase and to about 1 megabase,between from about 1 kilobase and to about 500 kilobases, between fromabout 10 kilobases and to about 1 megabases, between from about 10kilobase and to about 500 kilobases, between from about 10 kilobase andto about 100 kilobases, etc.).

Nucleic acid molecule assembled by methods of the invention may be, forexample, single stranded, partly single stranded or double stranded,closed, circular (e.g., a plasmid); nicked, circular; or linear (e.g., aplasmid, a chromosome, etc.). Further, methods of the invention may beperformed such that two or more (e.g., two, three, four, five, six, ten,twenty, etc.) assembled nucleic acid molecules are simultaneously formedin the same reaction mixture.

The invention further provides methods for producing product nucleicacid molecules. In some instances such the methods comprise:

(a) designing a product nucleic acid molecule of between 10 kilobasesand 500 kilobases in size (e.g., between 500 bases and 500 kilobases,between 500 bases and 100 kilobases, between 500 bases and 1 kilobase,between 500 bases and 800 bases between 2 kilobases and 100 kilobases,between 2 kilobases and 50 kilobases, between 2 kilobases and 5kilobases, between 10 kilobases and 500 kilobases, between 10 kilobasesand 300 kilobases, between 10 kilobases and 200 kilobases, between 10kilobases and 100 kilobases, between 10 kilobases and 50 kilobases,etc.), wherein the product nucleic acid molecule is defined bynucleotide sequence;

(b) synthesizing a plurality of individual nucleic acid molecules whichdiffer in nucleotide sequence, wherein each individual nucleic acidmolecule is synthesized to prepare a quantity of between 1,000 and1.0×10⁹ copies and wherein the individual nucleic acid molecules arecapable of hybridizing with one or more of the other individual nucleicacid molecules;

(c) combining the individual nucleic acid molecules synthesized in (b)under conditions which allow for hybridization of the individual nucleicacid molecules under conditions which allow for the formation of atleast one larger nucleic acid molecule; and

(d) combining the at least one larger nucleic acid molecule formed in(c) with one or more additional nucleic acid molecules to form theproduct nucleic acid molecule, wherein the product nucleic acid moleculecontains less than one sequence error per kilobase.

In many instances, an error correction process is employed duringgeneration of product nucleic acid molecules. One place in the abovework flow where an error correction process may be performed is afterstep (b). Error correction processes are described elsewhere herein andwill often include the use of one or more mis-match repair endonuclease.

The number of individual nucleic acid molecule synthesized as part ofthe preparation of product nucleic acid molecules may vary greatly butinclude between 1,000 and 1.0×10⁹ copies, between 1,000 and 1.0×10⁸copies, between 1,000 and 1.0×10⁷ copies, between 1,000 and 1.0×10⁶copies, between 1,000 and 1.0×10⁵ copies, between 2.0×10⁷ and 1.0×10⁹copies, between 5.0×10⁷ and 1.0×10⁹ copies, between 7.0×10⁷ and 1.0×10⁹copies, between 2.0×10⁷ and 8.0×10⁸ copies, between 2.0×10⁹ and 5.0×10⁸copies, between 5.0×10⁴ and 1.0×10⁹ copies, between 1.0×10⁶ and 1.0×10⁹copies, between 1.0×10⁷ and 1.0×10⁸ copies; etc.

In many instances, polymerase chain reactions may be used to amplify theat least one larger nucleic acid molecule formed in step (c) in theabove product nucleic acid molecule preparation processes.

Plate formats for the synthesis of nucleic acid molecules are describedelsewhere herein and they may be used in the above product nucleic acidmolecule preparation processes. Further, when individual nucleic acidmolecules are synthesized on beads, wherein each bead may be containedin a well. Further, beads used in this aspect of the invention, as wellas other aspects of the invention may be, for example of sizes such asbetween 1 μm and 100 μm in diameter, between 5 μm and 50 μm in diameter,between 3 μm and 100 μm in diameter, between 5 μm and 100 μm indiameter, between 20 μm and 100 μm in diameter, between 5 μm and 60 μmin diameter, between 10 μm and 100 μm in diameter, etc. In someembodiments beads may be of a size of about 30 μm in diameter (e.g.between 28 and 32 μm).

The invention also includes methods for producing nucleic acid moleculein small amounts and with high sequence fidelity. In some aspects, theinvention includes a method for generating a nucleic acid molecule, themethod comprising synthesizing the nucleic acid molecule in a totalamount of between 3.0×10⁶ and 4.0×10⁸ molecules, wherein the number ofsequence errors is between 1 in 100 to 1 in 500.

The invention thus includes methods for the generation of collections ofnucleic acid molecules, including methods comprising:

(a) synthesizing a plurality of nucleic acid molecules, wherein eachnucleic acid molecule is prepared in a microquantity;

(b) joining some or all of the nucleic acid molecules present in thepool formed in (b) to form a plurality of larger nucleic acid molecules;and

(c) assembling the plurality of larger nucleic acid molecules to formthe collection of nucleic acid molecules, wherein the collection ofnucleic acid molecules from bioinformatic information selected from thegroup consisting of:

(1) a copy DNA (cDNA) library containing only DNA corresponding tomessenger RNA (mRNA) molecules;

(2) a partial cDNA library containing DNA molecules corresponding toless than the full complement of mRNA molecules found in the cell typethat the bioinformatic information was derived from; and

(3) a collection of nucleic acid molecules in which some or all of thenucleic acid molecules are codon altered variants of nucleic acidmolecules found in the cell type that the bioinformatic information wasderived from.

The invention also provides method for the generation of selfreplicating nucleic acid molecules formed from smaller chemicallysynthesized nucleic acid molecules. In some embodiments, such method maycomprise one or more of the following steps:

(a) synthesizing a plurality of nucleic acid molecules, wherein eachnucleic acid molecule is prepared in a microquantity in a plate;

(b) joining some or all of the nucleic acid molecules present in thepool formed in (b) to form a plurality of larger nucleic acid molecules;and

(c) assembling the plurality of larger nucleic acid molecules to formthe self replicating nucleic acid molecule.

Self replicating nucleic acid molecules prepared by methods of theinvention include chromosomes, artificial chromosomes (such as, forexample, BACs or YACs), plasmids and genomes (e.g., genomes such asviral, nuclear, prokaryotic (e.g., bacterial, algal, etc.) chloroplast,or mitochondrial genomes).

The invention also includes methods for synthesizing and assemblingnucleic acid molecules which encode more than one expression product,the methods comprising:

(a) synthesizing a plurality of nucleic acid molecules, wherein eachnucleic acid molecule is prepared in a microquantity;

(b) joining some or all of the nucleic acid molecules present in thepool formed in (a) to form a plurality of larger nucleic acid molecules;and

(c) assembling the plurality of larger nucleic acid molecules to formthe nucleic acid molecules which encode more than one expressionproduct.

In various aspects of the invention, the more than one expressionproducts may be proteins involved in the same biological pathway. Inmore specific aspects, the more than one expression products may beproteins involved in the same biological pathway are enzymes thatcatalyze a series of chemical reactions in the biological pathway.Further, such chemical reactions in the same biological pathway may besequential reactions in the sense that one chemical reaction followsanother either directly (directly sequential) or after one or moreintervening reaction has occurred.

Biological pathway referred to herein include those that results in theproduction of an end product selected from the group consisting of (a)biofuel precursors; (b) antibiotics or antibiotic precursors; (c) foodcomponents; (d) a chemical intermediate (e.g., 1,4-butanediol,2,3-butanediol, benzene, butadiene, 2-butanol, 3-hydroxypropionic acid,acrylic acid, adipic acid, aminocaproic acid, caprolactam, acetylene,n-butanol, cyclohexanone, fumarate, 4-hydroxy butyrate, GBL/BDO,hexamethylenediamine, isobutanol, isopropanol, n-propanol, long chainalcohol, methacrylic acid/methyl methacrylate, methyl ethyl ketone,propylene, putrescine, muconic acid, p-toluate, terephthalic acid,acetic acid, glucaric acid); (d) industrial enzymes, and (e) naturalproducts. Biofuel precursors include alcohols selected from the groupconsisting of (a) butanol; (b) pentanol; (c) hexanol; (d) heptanol; and(e) octanol. Food components include livestock feed components,including amino acids selected from the group consisting of: (a)L-lysine; (b) L-threonine; (c) L-methionine; (d) L-leucine; (e)L-isoleucine: (f) L-valine, and (g) Homoserine.

Assembled nucleic acid molecules may be introduced into any number ofcells including prokaryotic and eukaryotic cell. Examples of such cellsinclude members of the genus Corynebacterium (e.g., Corynebacteriumglutamicum), Pseudomonas sp. (Pseudomonas aeruginosa), Saccharomycescerevisiae, Bacillus sp. (Bacillus lentus, Bacillus coagulans, Bacillussubtilis), Aspergillus sp. (Aspergillus terreus, A. niger, Aspergillusversicolorr), Streptomycetes spp. (Streptomyces griseus, StreptomycesViolaceans, Streptomyceshygroscopicus, Streptomyces octosporus),Clostridium(clostridia), Clostridium thermocellum, Clostridiumacetobutylicum, Clostridium beijerinckii, Clostridium butyricum,Clostridium jungdahlii, Clostridium aceticum, Clostridiumsaccharobutylicum, Clostridium saccharoperbutylacetonicum, Trichodermareesei (Hypocrea jecorina), Kluyveromyces (lactis), Neurospora crassa,Yarrowia lipolitica, Humicola (Humicola grisea), Hansenula polymorpha(Pichia angusta), Acetobacters, Zymomonas, Chrysosporium,Thermoanaerobacter, Pichia stipitis, Myxobacteria, Mortierellaisabellina, Actinobacillus succinogenes, Anaerobio spirillumsucciniciproducens, Pichia kudriavzevii/Issatchenkia orientalis (Yeast)(Candida krusei), Bifidobacterium, Bacillus coagulans GBI-30,Bifidobacterium animalis subsp. lactis BB-12, Bifidobacterium longumsubsp. infantis 35624, Lactobacillus acidophilus NCFM, Lactobacillusparacasei, Lactobacillus johnsonii La1, Lactobacillus plantarum,Lactobacillus reuteri, Saccharomvyces boulardii, Lactobacillusrhamnosus, Lactobacillus acidophilus NCFM, Bifidobacterium bifidumBB-12, Lactobacillus casei, Lactobacillus plantarum, Xanthomonas (X.campestris), Archea (Halobacterium sp. NRC-1, Sulfolobus tokodaii,Sulfolobus tokodaii Methanocaldococcus jannaschii, Thermoplasmaacidophilum and Thermoplasma volcanium), Rhodobacter sphaeroides,Ralstonia eutropha, Sporomusa species, Clostridium jungdahlii,Clostridium aceticum, Moorella thermoacetica, Geobacter species,Shewanella sp., Candida glabrata, Candida sonorensis, Candidatropicalis, Hansenula polymorpha, Issatchenkia orientalis, Kluyveromyceslactis, Kluyveromyvces marxianus, Kluyveromyces thermotolerans, Pichiastipidis, Saccharomyces bayaitus, Saccharomyces bulderi, Saccharomycesuvarum, Sachharomyces cerevisiae, Schizosaccharomyces pombe, YarrowiaLipolytica, Zygosaccharomyces bailii, Biodegredation (Aromatoleumaromaticum, Dechloromonas aromatica, Desulfitobacterium hafniense,Geobacter metallireducens, Alcanivorar borkumensis, Mycobacteriumtuberculosis), Deinococcus radiodurans, Actinoplanes regularis, Nocardiaorientalis, Actinocorrulia regularis, Tolypocladium inflatum, Monascusruber, Janibacter limonus, Actinomadura sp., Verucosispora sp., Muscodaralbus, and Neurospora crassa.

As one skilled in the art would understand, many aspects of theinvention are well suited for automation. Automated systems are oftendriven by software which may perform repetitive tasks, especially whenintegrated with hardware designed for micromanipulation of componentsand reagent flows. Thus, according to various embodiments describedherein, methods of assembling and synthesizing nucleic acids may beimplemented on a computing system. Further, according to variousembodiments described herein, processor-executable instructions forassembling and synthesizing nucleic acids. Thus, in some aspects theinvention includes non-transitory computer-readable storage mediaencoded with instructions, executable by a processor, for generatingassembled nucleic acid molecule, the instructions comprisinginstructions for:

(a) synthesizing a plurality of nucleic acid molecules, wherein eachnucleic acid molecule is prepared in a microquantity in the well of aplate;

(b) combining the nucleic acid molecules generated in (a) to produce apool;

(c) joining some or all of the nucleic acid molecules present in thepool formed in (b) to form a plurality of larger nucleic acid molecules;

(d) eliminating nucleic acid molecules which contain sequence errorsfrom the plurality of larger nucleic acid molecules formed in (c) toproduce an error corrected nucleic acid molecule pool; and

(e) assembling the nucleic acid molecules in the error corrected nucleicacid molecule pool to form the assembled nucleic acid molecule.

The invention also includes systems for generating assembled nucleicacid molecules, the system comprising:

a processor; and

a memory encoded with processor-executable instructions for:

(a) synthesizing a plurality of nucleic acid molecules, wherein eachnucleic acid molecule is prepared in a microquantity in the well of aplate;

(b) combining the nucleic acid molecules generated in (a) to produce apool;

(c) joining some or all of the nucleic acid molecules present in thepool formed in (b) to form a plurality of larger nucleic acid molecules;

(d) eliminating nucleic acid molecules which contain sequence errorsfrom the plurality of larger nucleic acid molecules formed in (c) toproduce an error corrected nucleic acid molecule pool; and

(e) assembling the nucleic acid molecules in the error corrected nucleicacid molecule pool to form the assembled nucleic acid molecule.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a general description of aspects of work flows of theinvention. The work flow is broken into four sections, referred to as“modules” for ease of description. The work flow on the right side ofthe figure shows some specific step included in some aspects of methodsof the invention.

FIGS. 2A-2B are schematic representations of a row of wells according toan embodiment of the invention. The darker area in well 1 indicates thepresence of a reagent (e.g., EGA) not present at a given time in theother wells.

FIG. 3 shows a nucleic acid assembly scheme. The thick ends on theassembled nucleic acid molecule shown at the bottom of the figurerepresent regions added by external primers, also referred to asterminal primers.

FIG. 4 shows a second nucleic acid assembly scheme. Dotted lines witharrows show PCR based synthesis direction and area.

FIG. 5 shows the assembly of two DNA fragments that do not share anyhomology into a vector using stitching nucleic acid molecules. The 69base pair double-stranded stitching nucleic acid molecules, shown inbold in the lower portion of the figure, share 30-bp homology with eachadjacent fragment (Fragments 1 and 2). These stitching nucleic acidmolecules are used to insert 9 bp at the junction of the adjacentfragments. The insertion bases are shown underlined.

FIG. 6 is a flow chart of an exemplary process for synthesis oferror-minimized nucleic acid molecules.

FIG. 7 is a work flow chart of an exemplary process for synthesis oferror-minimized nucleic acid molecules. Different strands of adouble-stranded nucleic acid molecule are represented by thicker andthinner line. “MME” refers to mis-match endonuclease. Small circlesrepresent sequence errors.

FIG. 8 generally illustrates methods for assembly and cloning of nucleicacid segments in yeast. In some embodiments of the invention, a numberof nucleic acid segments (one of which is a vector) are co-transformingthe fragments into a yeast host cell, where they are assembled byhomologous recombination to form, for example, a closed, circularnucleic acid molecule.

FIG. 9 is a drawing of an electrical coil that may be used in thepractice of the invention.

FIG. 10 is cross-sectional view of one embodiment of a fluid reagentdelivery system suitable for use with the invention.

FIG. 11 shows a library of linear, nucleic acid molecules (top)generated by methods of the invention and a vector (bottom) designed toaccept library members. The upper portion of the figure shows a seriesof lines representing four members of the library. The lower opencircular line represents a vector. The blocks on each end of the nucleicacid molecule represent nucleic acid segments which facilitate joining(e.g., GATEWAY® sites, regions of homology, etc.). The numbers and thetermini of the nucleic acid molecules indicate compatible ends.

FIGS. 12A and 12B show a series of variant nucleic acid molecules thatmay be prepared by methods of the invention and their encoded amino acidsequences. FIG. 12A shows variant nucleic acid molecules that encodedifferent amino acid sequences. FIG. 12B shows variant nucleic acidmolecules that use different codons but encode the same amino acidsequence.

FIG. 13 shows two different fluid removal options for microwell plateembodiments of synthesis platforms.

FIGS. 14A and 14B show two different views of a nucleic acid moleculessynthesis platform designed to generate identical nucleic acid moleculesin each row 1401. FIG. 14A is a top view and FIG. 14B is a side view.Shown in the figure are fluidic channels 1401, two electrodes associatedwith each channel/row of wells 1402 and a series of wells containingnucleic acid synthesis substrates (e.g., individual beads) located inwells 1400. In some embodiments, the wells will be spaced 300 μm apartand will be cylindrical in shape with a diameter of 40 μm and a depth of35 μm.

FIG. 15 is a block diagram that illustrates a computing system, uponwhich embodiments of the present teachings may be implemented.

FIG. 16 is a schematic of automated system for performing methods of theinvention.

FIG. 17 is a top view schematic of a channel “chip”.

DETAILED DESCRIPTION OF THE INVENTION Definitions

Solid Support: As used herein, the term solid support refers to a porousor non-porous material on which polymers such as nucleic acid moleculescan be synthesized and/or immobilized. As used herein “porous” meansthat the material contains pores which may be of non-uniform or uniformdiameters (for example in the nm range). Porous materials include paper,synthetic filters etc. In such porous materials, the reaction may takeplace within the pores. The solid support can have any one of a numberof shapes, such as pin, strip, plate, disk, rod, fiber, bends,cylindrical structure, planar surface, concave or convex surface or acapillary or column. The solid support can be a particle, includingbead, microparticles, nanoparticles and the like. The solid support canbe a non-bead type particle (e.g., a filament) of similar size. Thesupport can have variable widths and sizes. For example, sizes of a bead(e.g., a magnetic bead) which may be used in the practice of theinvention are described elsewhere herein. The support can be hydrophilicor capable of being rendered hydrophilic and includes inorganic powderssuch as silica, magnesium sulfate, and alumina; natural polymericmaterials, particularly cellulosic materials and materials derived fromcellulose, such as fiber containing papers such as filter paper,chromatographic paper or the like.

In some embodiments, solid support may be fragmentable. Solid supportsmay be synthetic or modified naturally occurring polymers, such asnitrocellulose, carbon, cellulose acetate, polyvinyl chloride,polyacrylamide, cross linked dextran, agarose, polyacrylate,polyethylene, polypropylene, poly (4-methylbutene), polystyrene,polymethacrylate, poly(ethylene terephthalate), nylon, poly(vinylbutyrate), polyvinylidene difluoride (PVDF) membrane, glass, controlledpore glass, magnetic controlled pore glass, magnetic beads, ceramics,metals, and the like; either used by themselves or in conjunction withother materials.

In some embodiments, the support can be in a chip, array, microarray ormicrowell plate format. In many instances, a support generated bymethods of the invention will be one where individual nucleic acidmolecules are synthesized on separate or discrete areas to generatefeatures (i.e., locations containing individual nucleic acid molecules)on the support.

In some embodiments, the size of the defined feature is chosen to allowformation of a microvolume droplet or reaction volume on the feature,each droplet or reaction volume being kept separate from each other. Asdescribed herein, features are typically, but need not be, separated byinterfeature spaces to ensure that droplets or reaction volumes orbetween two adjacent features do not merge. Interfeatures will typicallynot carry any nucleic acid molecules on their surface and willcorrespond to inert space. In some embodiments, features andinterfeatures may differ in their hydrophilicity or hydrophobicityproperties. In some embodiments, features and interfeatures may comprisea modifier. In one embodiment of the invention the feature is a well ormicrowell or a notch.

Nucleic acid molecules may be covalently or non-covalently attached tothe surface or deposited on the surface.

In one embodiment of the invention, Module 1 can involve the use of morethan one solid support. In some embodiments, two or more solid supportsmay be arranged on a plate. Any arrangement of the solid supports couldbe employed such as rows or columns or a combination thereof. Forexample, rows can be aligned and/or the columns can be aligned. In otherembodiments, rows and/or columns are equally spaced and staggered.Spacing between rows and/or between columns can be variable. The numberof the solid supports comprised in, for example, a plate may bevariable. In some embodiments, a plate may contain up to 1536 (or more)solid supports.

Nucleic Acid Molecule: As used herein the term “nucleic acid molecule”refers to a covalently linked sequence of nucleotides or bases (e.g.,ribonucleotides for RNA and deoxyribonucleotides for DNA but alsoinclude DNA/RNA hydrids where the DNA is in separate strands or in thesame strands) in which the 3′ position of the pentose of one nucleotideis joined by a phosphodiester linkage to the 5′ position of the pentoseof the next nucleotide. Nucleic acid molecule may be single- ordouble-stranded or partially double-stranded. Nucleic acid molecule mayappear in linear or circularized form in a supercoiled or relaxedformation with blunt or sticky ends and may contain “nicks”. Nucleicacid molecule may be composed of completely complementary single strandsor of partially complementary single strands forming at least onemismatch of bases. Nucleic acid molecule may further comprise twoself-complementary sequences that may form a double-stranded stemregion, optionally separated at one end by a loop sequence. The tworegions of nucleic acid molecule which comprise the double-stranded stemregion are substantially complementary to each other, resulting inself-hybridization. However, the stem can include one or moremismatches, insertions or deletions.

Nucleic acid molecules may comprise chemically, enzymatically, ormetabolically modified forms of nucleic acid molecules or combinationsthereof. Chemically synthesized nucleic acid molecules may refer tonucleic acids typically less than or equal to 150 nucleotides long(e.g., between 5 and 150, between 10 and 100, between 15 and 50nucleotides in length) whereas enzymatically synthesized nucleic acidmolecules may encompass smaller as well as larger nucleic acid moleculesas described elsewhere in the application. Enzymatic synthesis ofnucleic acid molecules may include stepwise processes using enzymes suchas polymerases, ligases, exonucleases, endonucleases or the like or acombination thereof. Thus, the invention provides, in part, compositionsand combined methods relating to the enzymatic assembly of chemicallysynthesized nucleic acid molecules.

Nucleic acid molecule also refers to short nucleic acid molecules, oftenreferred to as, for example, primers or probes. Primers are oftenreferred to as single-stranded starter nucleic acid molecules forenzymatic assembly reactions whereas probes may be typically used todetect at least partially complementary nucleic acid molecules. Anucleic acid molecule has a “5′-terminus” and a “3′-terminus” becausenucleic acid molecule phosphodiester linkages occur between the 5′carbon and 3′ carbon of the pentose ring of the substituentmononucleotides. The end of a nucleic acid molecule at which a newlinkage would be to a 5′ carbon is its 5′ terminal nucleotide. The endof a nucleic acid molecule at which a new linkage would be to a 3′carbon is its 3′ terminal nucleotide. A terminal nucleotide or base, asused herein, is the nucleotide at the end position of the 3′- or5′-terminus. A nucleic acid molecule sequence, even if internal to alarger nucleic acid molecule (e.g., a sequence region within a nucleicacid molecule), also can be said to have 5′- and 3′-ends.

Overview:

The invention relates, in part, to compositions and methods for thepreparation of nucleic acid molecules. While the invention has numerousaspects and variations associated with it, some of these aspects andvariations are set out in FIG. 1 in outline form.

One advantage of the invention is that for, many applications, smallamounts of synthesized nucleic acid are suitable for achieving anintended purpose (e.g., preparation of microarrays, construction of aplasmid which contains a selectable marker, etc.). In some instances,small amounts of nucleic acid are suitable for working with due tofactors such as enzymatic (e.g., PCR) and intracellular amplification.

The left side of FIG. 1 shows four general “modules” representingdifferent portions of some embodiments of the invention. Thus, in someaspects, the invention involves one or more of the following: (1)nucleic acid molecule synthesis, (2) pooling of nucleic acid molecules,(3) assembly of a plurality of nucleic acid molecules, and/or (4)transfer of assembled nucleic acids (e.g., transfer to a cell).

In relation to more specific embodiments of the invention, the rightside of FIG. 1 shows some additional details related to the modulesshown on the left side of the figure. Above a number of the text blocksare bolded terms such as “ENZYMATIC” and “CELLULAR”. These termsindicate exemplary general means by which the process referred to can beperformed. As one skilled in the art would understand, some processescan be performed, for example, either chemically, enzymatically, or in acell.

Module 1, as shown in FIG. 1 refers to a single process termed“Microscale Parallel Nucleic Acid Molecule Synthesis”. As set outelsewhere herein, this process will typically involve several stepswhich will vary with how the process is performed. In many embodiments,the general function of Module 1 will be the generation of a pluralityof nucleic acid molecules. These nucleic acid molecules may be designedas a group to be joined to form one or more larger nucleic acid moleculeor when contacted with additional nucleic acid molecules (e.g.,“stitching” nucleic acid molecules).

Module 2, as shown in FIG. 1 refers to processes termed “Pooling ofSolid Supports”, “Nucleic Acid Molecule Cleavage”, and “Deprotection”.The general function of Module 2 will be the preparation of nucleic acidmolecules for participation in one or more process referred to in Module3. This will often mean combining nucleic acid molecules which differ insequence and the removal of any chemical groups which are either notnecessary or not desirable for the performance of one or more processesreferred to in Module 3.

Using Module 2 as an example, as one skilled in the art would recognize,FIG. 1 shows general embodiments of the invention. More specifically,Module 2 refers to the pooling of solid supports. These supports willtypically contain nucleic acid molecules. In some embodiments, nucleicacid molecules may be obtained in a form free of solid supports, thenpooled.

Module 3, as shown in FIG. 1 refers to the processes termed “FragmentAmplification and Assembly”, “Error Correction”, and “Final Assembly”.The general function of Module 3 processes is the generation ofassembled nucleic acid molecules with high sequence fidelity, withcomparison to the sequence of nucleic acid molecules which were soughtto be produced.

Module 4, as shown in FIG. 1 refers to the processes of termed“Recipient Cell Insertion”. As one skilled in the art would understand,introduction of nucleic acid molecules generated by methods of theinvention into cells is only one application. In most instances, anucleic acid molecule assembled according to methods of the inventionwill be designed for a specific application. Applications vary widelyand include biofuel production, bioremediation, and chemical precursorproduction.

In some embodiments, amino group containing support matrix having apolyvinyl backbone may be used as solid support. For example,monodispersed particles obtained by methods as described in U.S. Pat.No. 6,335,438 the disclosure of which is incorporated herein byreference, may be used in the practice of the invention.

Module 1

In the invention, the nucleic acid molecules may be attached to solidsupports, such as particles or beads (e.g., controlled pore glassbeads). In one embodiment, magnetic microbeads are used as solidsupports. In many instances, single-activated porous 1 μm sizemicrobeads with large surface to volume ratios may be used in thecurrent invention. The uniform nature of such monodispersed particlesgenerally provides for uniform reaction rates particularly suited tosynthesis in automated chemical synthesizers (e.g., nucleic acidmolecule synthesizers). Beads may initially be provided with a reactivegroup. For example, in some embodiments of the invention, DYNABEADS®M-280 (Dynal Biotech ASA, Oslo, Norway) may be used. DYNABEADS® M-280are 2.8 μm beads which come in a number of forms. M-280 beads tend arefairly uniform, superparamagnetic, polystyrene beads coated with apolyurethane layer. These beads may be obtained with various chemicalactivation groups suitable for use for different applications.

Magnetic bead technology is described in U.S. Pat. No. 5,512,439, whichis incorporated herein by reference.

Synthesis substrates other than those composed of CPG or magneticmaterials may also be used with the invention and include those composedof polystyrene (e.g., polystyrene-1% divinylbenzene, macroporouspolystyrene, and poly(ethylene glycol)-polystyrene (PEG-PS)), polyamide(e.g., polyamide bonded silica gel), silica gel, and cellulose. Some ofthese substrates are available in resin form. In many instances,substrates that are resins may be placed in wells, instead of or inconjunction with beads, and may be used for nucleic acid synthesis.

Other nucleic acid ligation methods, and arrays which employ them, areknow in the art. For example, methods are known which use an amine or aperoxide (which opens to an ether bridge) activated surface. As notedelsewhere herein, for EGA methods in the art, a hydroxyl group has beendescribed and used to link nucleic acid to a silica magnetic beadsurface. The invention includes such linking methods and compositionswhich contain them.

In some instances, it may also be desired to use a semi-solid supportthat may have a gel-like or viscous consistence or matrix instead of asolid support. The invention contemplates this and in suitable instanceshere where a solid support is referred to a non-solid support may beused.

Factors which determine the amount of nucleic acid which can besynthesized include surface area and size of particles upon whichsynthesis occurs. Thus, to some extent, support (e.g., bead) parameterscan be adjusted to alter the amount of nucleic acid synthesized. Beadswhich may be used in the practice of the invention may vary widely interms of size, including the following size ranges: from about 0.01 m toabout 1,000 μm, from about 0.1 μm to about 1,000 μm, from about 1.0 μmto about 1,000 μm, from about 0.01 μm to about 400 μm, from about 0.01μm to about 200 μm, from about 0.01 μm to about 100 μm, from about 0.1μm to about 100 μm, from about 0.1 μm to about 50 μm, from about 1.0 μmto about 600 μm, from about 1.0 μm to about 400 μm, from about 1.0 μm toabout 200 μm, from about 1.0 μm to about 100 μm, from about 2.0 μm toabout 400 μm, from about 2.0 μm to about 200 μm, from about 5.0 μm toabout 500 μm, etc. in average diameter.

Further, beads may be used which allow for an average amount of nucleicacid to be produced in the following amounts: from about 0.001 nanomolesto about 1,000 nanomoles, from about 0.1 nanomoles to about 1,000nanomoles, from about 1.0 nanomole to about 1,000 nanomoles, from about5.0 nanomoles to about 1,000 nanomoles, from about 10 nanomoles to about1,000 nanomoles, from about 30 nanomoles to about 1,000 nanomoles, fromabout 50 nanomoles to about 1,000 nanomoles, from about 200 nanomoles toabout 1,000 nanomoles, from about 1.0 nanomole to about 500 nanomoles,from about 1.0 nanomole to about 250 nanomoles, from about 10 nanomolesto about 500 nanomoles, etc.

TABLE 1 Number of Nucleic Acid Molecules Nucleic Acid (Nanomoles) 1.26 ×10⁵ 2.09 × 10⁻¹⁰ 3.14 × 10⁶ 5.22 × 10⁻⁰⁹ 1.26 × 10⁷ 2.09 × 10⁻⁰⁸ 1.13 ×10⁸ 1.88 × 10⁻⁰⁷ 3.14 × 10⁸ 5.22 × 10⁻⁰⁷ 1.26 × 10⁹ 2.09 × 10⁻⁰⁶

In many instances, the yield of nucleic acid molecules chemicallysynthesized decreases once a certain size has been reached. In manyembodiments of the invention, chemically synthesized nucleic acidmolecules will be in the range of from about 8 to about 100 nucleotides,from about 8 to about 35 nucleotides, from about 8 to about 40nucleotides, from about 8 to about 50 nucleotides, from about 8 to about100 nucleotides, from about 15 to about 100 nucleotides, from about 15to about 75 nucleotides, from about 15 to about 50 nucleotides, fromabout 20 to about 60 nucleotides, from about 40 to about 400nucleotides, from about 40 to about 300 nucleotides, from about 40 toabout 200 nucleotides, from about 40 to about 100 nucleotides, fromabout 40 to about 90 nucleotides, from about 50 to about 400nucleotides, from about 50 to about 300 nucleotides, from about 50 toabout 200 nucleotides, from about 50 to about 100 nucleotides, fromabout 50 to about 90 nucleotides, from about 50 to about 80 nucleotides,from about 75 to about 400 nucleotides, from about 75 to about 300nucleotides, or from about 75 to about 200 nucleotides.

As one skilled in the art would recognize, the amount of nucleic acidrequired to be produced will vary with, for examples, the applicationand the efficiency of assembly methods used. When a replicable molecule(e.g., via PCR, insertion into a cell, etc.) is generated, theoreticallyonly one assembled nucleic acid molecule need be generated. If thenumber of nucleic acid molecules generated are reduced to the pointwhere theoretically only one fully assembled nucleic acid molecule isgenerated, then half the time no fully assembled nucleic acid moleculewill generated. Thus, one lower limit for the amount of nucleic acid tobe produced using methods of the invention is based upon the number offully assembled nucleic acid molecules which may be generated. Thisnumber will often vary with the number of synthetic nucleic acidmolecules that must be combined to form the final construct. Methods ofthe invention will typically be designed to generate from about 1 toabout 500,000, from about 10 to about 500,000, from about 100 to about500,000, from about 500 to about 500,000, from about 1 to about 1,000,from about 1 to about 500, from about 10 to about 1,000, from about 10to about 500, from about 100 to about 1,000, from about 100 to about500, from about 100 to about 5,000, from about 100 to about 50,000, fromabout 100 to about 250,000, from about 1,000 to about 50,000, etc.assembled nucleic acid molecules.

As one skilled in the art would understand, nucleic acid synthesissubstrate area directly reflects the number of nucleic acid moleculeswhich may be synthesized on that substrate. Table 2 below shows beadsize, surface area calculations and an estimated number of nucleic acidmolecules that may be generated on the specified beads.

TABLE 2 Bead Diam. (μm) Surface Area (μm²) No. of Molecules 1 314.2 1.26× 10⁵ 5 7,855 3.14 × 10⁶ 10 31,416 1.26 × 10⁷ 30 282,743 1.13 × 10⁸ 50785,398 3.14 × 10⁸ 100 3,141,593 1.26 × 10⁹ Note: The effective surfacearea for the beads used to generate the above data is estimated to be100 times higher than the spherical surface are.

In some embodiments, oligonucleotide synthesis will be performed using2.8 μm beads in a plate with one bead per well. Further, the wells maybe designed as cylindrical holes or chambers that are 4 μm and 3 μmdeep. When well spacing of 100 μm is used, a 10 mm² chip can accommodate10,000 wells. In many instances when plates are made by etching, thewells will be of a non-cylindrical shapes and may be pyramid, cone orquadratic shaped. In some instances, the wells may be in the shape of areverse, truncated cone.

The number of individual nucleic acid molecules generated will also varywith the application. While costs savings can be achieved by reagentusage reductions, it will generally be desirable to generate enoughnucleic acid molecules need for, for example, efficient assembly.Further, the number of nucleic acid molecules having a particularnucleotide sequence produced with generally reflect the “carryingcapacity” of the synthesis substrate. For example, a 30 micron beadtypically can be used to generate about 1,000,000 nucleic acidmolecules. For example, in many instances, as bead size, decreases, sowill the number of nucleic acid molecules that may be produced on eachbead.

Methods of the invention may be used to generate from about 100 to about20,000,000, from about 1,000 to about 20,000,000, from about 10,000 toabout 20,000,000, from about 100 to about 5,000,000, from about 1,000 toabout 5,000,000, from about 10,000 to about 5,000,000, from about 100 toabout 1,000,000, from about 1,000 to about 1,000,000, from about 10,000to about 10,000,000, from about 100 to about 500,000, from about 1,000to about 500,000, from about 10,000 to about 500,000, etc. nucleic acidmolecules designed to have the same nucleotide sequence.

The number of nucleic acid molecule synthesis sites (e.g., wells) canvary greatly and will be determined by a number of factors including (1)the limitations of engineering and nucleic acid molecule synthesishardware and (2) the amount of nucleic acid which is desired (seeelsewhere herein for a discussion of this factor). As examples, thenumber of nucleic acid molecule synthesis sites (e.g., wells) insynthesis platforms used in the practice of the invention may vary intotal number between 9 and 200,000, between 9 and 100,000, between 9 and20,000, between 9 and 1,000, between 9 and 500, between 1,000 and200,000, between 1,000 and 400,000, between 1,000 and 500,000, between1,000 and 1,00,000, between 1,000 and 10,000,000, between 20,000 and1,000,000, between 50,000 and 10,000,000, between 10,000 and 5,000,000,between 1,000 and 100,000, between 2,000 and 100,000, between 5,000 and100,000, between 10,000 and 100,000, between 20,000 and 100,000, between30,000 and 100,000, between 1,000 and 80,000, between 1,000 and 70,000,between 1,000 and 50,000, between 1,000 and 40,000, between 1,000 and30,000, between 1,000 and 20,000, between 1,000 and 10,000, between1,000 and 8,000, between 1,000 and 5,000, between 5,000 and 50,000,between 10,000 and 50,000, between 5,000 and 35,000, etc. In addition,the number of nucleic acid molecule synthesis sites (e.g., wells) mayvary between 1,000 and 5,000, between 1,000 and 10,000, between 1,000and 20,000, between 1,000 and 30,000, between 2,000 and 5,000, between2,000 and 10,000, between 4,000 and 15,000, between 100 and 1,000,between 100 and 3,000, between 100 and 5,000, between 250 and 5,000,etc. per mm².

The amount of reagent space per nucleic acid molecule synthesis site(e.g., well) will vary with the size and shape of the well and, inparticular, the area of the space capable of accepting reagents. Thiswill vary with factors such as whether the nucleic acid moleculesynthesis site is a flat surface (e.g., relying on surface tension tokeep reagents localized over the synthesis site or a cavity (e.g., awell). Also, the amount of reagent applied may be determined by theamount of reagent necessary to cover the synthesis site, deliver thenecessary amount of reactant(s), and/or dilute, remove, or wash awayreagents present at the synthesis site. The amount of reagent applied(when the reagent is a liquid) and the amount of reagent space at thesynthesis site may vary greatly including between 0.001×10⁻¹⁵ l(femtoliter) and 100 μl, between 0.01×10⁻¹⁵ l (femtoliter) and 100 μl,between 0.1×10⁻¹⁵ l (femtoliter) and 100 μl, between 1.0×10⁻¹⁵ l(femtoliter) and 100 μl, between 0.1×10⁻¹⁵ l (femtoliter) and 1 μl,between 0.1×10⁻¹⁵ l (femtoliter) and 500 nl, between 0.1×10⁻¹⁵ l(femtoliter) and 100 nl, between 0.1×10⁻¹⁵ l (femtoliter) and 1 nl,between 0.1×10⁻¹⁵ l (femtoliter) and 500 pl (picoliter), between0.1×10⁻¹⁵ l (femtoliter) and 100 pl, between 0.1×10⁻¹⁵ l (femtoliter)and 10 pl, between 0.1×10⁻¹⁵ l (femtoliter) and 1 pl, between0.001×10⁻¹⁵ l (femtoliter) and 1 pl, between 0.001×10⁻¹⁵ l (femtoliter)and 1.0×10⁻¹⁵ l (femtoliter), between 0.001×10⁻¹⁵ l (femtoliter) and100×10⁻¹⁵ l (femtoliter), between 1.0×10⁻¹⁵ l (femtoliter) and 500×10⁻¹⁵l (femtoliter), etc.

To make the solid support material suitable for nucleic acid moleculesynthesis, non-nucleosidic linkers or nucleoside succinates may becovalently attached to reactive amino groups. If necessary, however,other surface functions such as carboxyl could be used to attach alinker carrying a hydroxyl group or alternatively a 3′-attachednucleotide.

The linker, when present, may be a chemical entity that attaches the3′-O of the nucleic acid molecule to the solid support (e.g., afunctional group on a solid support). In most cases, the linker will bestable to all the reagents used during nucleic acid molecule synthesis,but cleavable under specific conditions at the end of the synthesisprocess. One linker commonly used in nucleic acid molecule synthesis isthe succinyl linker. Different linkers with different properties areknown to those skilled in the art and can be selected by the skilledperson depending on the downstream process requirements.

Nucleosidic solid supports (e.g., support prederivatized with base) arewidely used in nucleic acid molecule synthesis. One example of such asupport is one where the 3′-hydroxy group of the 3′-terminal nucleosideresidue is attached to the solid support via a 3′-O-succinyl arm. Theuse of nucleosidic solid supports requires usage of different types ofbeads (one for each base). However, the fact that a nucleosidic solidsupport has to be selected in a sequence-specific manner (according tothe first base required for each nucleic acid molecule) reduces thethroughput of the entire synthesis process due to laboriouspre-selection and distribution of beads attached to a specific starterbase to individual microwells.

A more convenient method for synthesis starts with a universal supportwhere a non-nucleosidic linker is attached to the solid supportmaterial. An advantage of this approach is that the same solid supportmay be used irrespectively of the sequence of the nucleic acid moleculeto be synthesized. One example of a universal support that can be usedin the current invention is described in U.S. Pat. No. 7,202,264, thedisclosure of which is incorporated herein by reference. However, otheruniversal linkers known by the skilled in the art may be equallyappropriate to carry out the invention. For the complete removal of thelinker and the 3′-terminal phosphate from the assembled nucleic acidmolecule, some of the universal solid supports known in the art requiregaseous ammonia, aqueous ammonium hydroxide, aqueous methylamine or amixture thereof.

A number of methods for synthesizing nucleic acid are known. Many ofthese methods follow a series of basic steps, such as, for example, thefollowing, with appropriate washing steps using, for example,acetonitrile, ethylacetate or other washing reagents suitable forpracticing the invention:

a) the first nucleotide, which has been protected at the 5′ position, isderivatized to a solid support, usually controlled pore glass (CPG), oris obtained prederivatized;

b) the sugar group of the first nucleotide is deprotected (e.g., viadetritlyation) (a process often referred to as “Deprotection”), using,for example, tricholoracetic acid in methylene chloride, which resultsin a colored product which may be monitored for reaction progress;

c) the second nucleotide, which has the phosphorus, sugar and basegroups protected, is added to the growing chain, usually in the presenceof a catalyst, such as, for example, tetrazole or 4,5-dicyanoimidazole(a process often referred to as “Coupling”);

d) unreacted first nucleotide is capped to avoid accumulation ofdeletions, using, for example, acetic anhydride and N-methylimidazole (aprocess often referred to as “Capping”);

e) the phosphite triester is oxidized to form the more stable phosphatetriester, usually using, for example, iodine reagents (a process oftenreferred to “Oxidizing”);

f) the process is repeated as needed depending on the desired length ofthe nucleic acid molecule; and

g) cleavage from the solid support is done, usually using aqueous orgaseous ammonia at elevated temperatures. The skilled in the art willrecognize that in certain embodiments of the invention the order ofsteps may vary or some of the steps including the washing steps may berepeated as appropriate according to the used protocol.

In the current invention, the state of the art phosphoramidite synthesischemistry is further improved by modification of specific steps of theabove protocol. In one embodiment organocatalysts can be used toimprove, for example, the efficiency of the coupling step.Organocatalysts and some uses of such catalysts are set out in Avenierand Hollfelder, Combining Medium Effects and Cofactor Catalysis:Metal-Coordinated Synzymes Accelerate Phosphate Transfer by 10⁸ Chem.Eur. J. 15:12371-12380 (2009) and Jordan et al., Asymmetricphosphorylation through catalytic P(III) phosphoramidite transfer:Enantioselective synthesis of D-myo-inositol-6-phosphate, Proc. Nat.Acad. Sci. USA, 107: 20620-20624 (2010).

In some embodiments, the invention makes use of localized chemicalreactions through the production of electrochemically generated acid(EGA). As an example, addressable electrical signals may be used for theproduction of acid at sufficient concentration to allow deprotection ofthe dimethoxytrityl (DMT) protecting group from surface. (Maurer et al.,“Electrochemically Generated Acid and Its Containment to 100 MicronReaction Areas for the Production of DNA Microarrays” PLoS, Issue 1, e34(December 2006).)

One issue with the production of EGA as part of a nucleic acid moleculesynthesis protocol on a surface (e.g., a microsurface) is “splash over”to adjoining regions. “Splash over”, which includes diffusion, canresult in reactions occurring in unintended location (e.g., caused bydiffusion of EGA). While such effects may be fairly minor when onereaction occurs, when multiple reactions occur in succession splash overeffects multiple reaction cycles may result in numerous misincorporatedbases. This issue can be addressed in several ways. One way is tooverlay the reaction areas with a buffer (e.g., a buffer containing anorganic base) which sufficiently neutralizes the acid if it moves fromthe local environment. Another way is through physical containment orcompartmentalization. For example, if the EGA is generated in a well andcatalyzes a reaction in that well, the well may be of sufficient size toprevent the acid from exiting. Containment within the well is thus afactor of the size of the well and the amount of acid generated. In somereaction formats, some acid will invariably exit the well. This shouldpose no problems unless a quantity sufficient to catalyze a reactionreaches another well in which that reaction is not supposed to occur. Asnoted above, the use of an overlaying buffer can be used to minimizesuch reactions.

Plates which may be used in the practice of the present inventioninclude modified forms of plates described in U.S. Patent PublicationNo. 2010/0137143 A1, the disclosure of which is incorporated herein byreference, shows such a representative plate format.

FIGS. 2A-2B are schematic representations of a row of wells 200according to an embodiment of the invention. The embodiment of FIGS.2A-2B illustrate five wells each containing a magnetic bead 201 at thebottom. Beneath each well is an electrode 202 which can deliver currentto the well that it is associated with. Each electrode is communicateswith a current controller 203 which regulates current to the electrode.The magnetic bead may contain a linker associated with an initialbuilding block. As an example, the bead may contain first nucleotide(with an A, T, C, G or U base, or a modified base, depending on thefirst base desired in the nucleic acid molecule to be synthesized). Thefirst base may be added as part of the synthesis process (e.g., with thebead having a protected hydroxyl group) or may be prederivatized priorto insertion into the well. In either event, in most cases, a protectivegroup will be present (e.g., at the 5′ position) which must be removedbefore another base may be covalently connected as part of a nucleicacid molecule chain.

Microfluidic channels (not shown in FIG. 2A) may be included forefficiently addition and removal of reagents from the wells. Thus, theinvention includes, in part, a microfluidic plate designed to interfacewith a microfluidic system for adding and removing fluids from wells ofthe plate. Microfluidic channels used in similar plates are described inU.S. Patent Publication No. 2010/0137143 A1, the disclosure of which isincorporated herein by reference for background information.

The cover of the plate 204 shown in FIG. 2A contains aligned electrodeswhich are connected to the current controller. A larger electrode (e.g.,an electrode which extends over the tops of all of the wells) may beincluded in the cover to “close the circuit”. Thus, the cover maycontain one electrode aligned with each well for which anelectrochemical reaction is sought to be, one electrode in operableconnection with all wells of the plate, or multiple electrodes some orall of which are in operable connection with two or more wells. In analternative embodiment, the cover electrode for each well is replacedwith one or more electrodes embedded or positioned along one or moresidewall of each well. Thus, it is not critical that electrodes bepositioned in the cover. In fact, in many instances, it will bedesirable (e.g., ease of manufacturing) to place the electrodes in aplace other than the cover.

Reference electrodes (RE) 205 may also be included to provide a stableand pre-defined electric potential. To apply a specific potential on aworking electrode (WE), the potential of the WE against the potential ofthe RE may be measured. Next the potential between counter electrode(CE) and WE may be adjusted until the potential between RE and CE hasthe correct value.

One method for deprotection may employ the oxidization of hydroquinoneto benzoquinone (redox system) on the WE in order to produce protons. Toset a specific pH in a well, a constant current may be applied for aspecified period of time. In instances of a less active WE, a strongincrease of the WE potential will occur. This can lead to unintendedreactions (e.g., oxidation of the solvent or damage of WE material athigh potential). To avoid this effect, the potential of the WE may becontrolled.

The current controller (interchangeably, controller) may be amicroprocessor or processor, such as shown in FIG. 15, for example Thecontroller (not shown) may comprise a conventional current controlsystem, including, for example, a microprocessor circuit incommunication with a memory circuit. The memory circuit may includeinstructions for directing the microprocessor circuit to energize one ormore of the electrodes (e.g., energize electrodes associated with well 1or a plurality of wells). Optionally, the memory circuit may includeinstructions for activating one of a pair of electrodes (e.g., activatethe bottom electrode associated with well 1). In still anotherembodiment, the memory circuit may include instructions for graduallyincreasing/decreasing bias to the electrodes so as to reduce possibilityof a sudden surge at the well.

In another embodiment, the current controller communicates with externalprocessor circuit(s) such as a potentiostat circuit, input/output(“I/O”) devices and displays. The circuit or circuit board enables thecontrol of the device and may also be used to communicate with otherdevices (such as PC, iPad, etc.).

In a variation of the embodiment of FIG. 2A, both electrodes (the anodeand the cathode) may be placed at the bottom of the well. This allowsfor electrical current to be generated near the bottom of the well,thereby generating a localized EGA in the area closely adjacent thebead. Depending on the method by which reagents are added to and/orremoved from the wells and other factors, such configuration can be usedto limit cross-talk between the wells, interference or unintended EGAcontamination.

A related embodiment is shown in FIG. 2B. Here the cover containsaligned electrodes 205 which extends into the reagent portion of thewell. Drainage tubes 206 are positioned at the bottom of each well.These drainage tubes serve several functions. One function is removingreagents at the completion of a chemical reaction step (e.g., baseaddition, washing, deprotection, etc.). Another function is lowering thefluid level for the deprotection step. In other words, fluid may beadded to all of the wells, then the fluid level may be lowered throughdrainage tubes before biasing the wells. Lowering the well's fluid levelreduces cross-spillage between wells and increases synthesis fidelity.The lowered fluid level also decreases potential cross-talk andcontamination between adjacent wells. The same is true of general fluidremoval through the bottle of the well. This is so because cross-wellcontamination with EGA can result in incorrect base incorporation. EvenEGA generated base mis-incorporation occurs in 0.5% of nucleic acidmolecules being synthesized in adjoining wells, the net result could beroughly a doubling of base mis-incorporation. Thus, drawing down thefluid level in the wells and bottom of the well drainage results inincrease synthesis fidelity.

One means for removing fluid from wells from the top of the wells. Thiscan be done by any number of means including the use of pipette tips orthe introduction of an absorbent material. In either instance, the goalwould be to remove enough fluid from each well to minimize “splashover”. In some instance, the only wells that fluid levels will bereduced in will be ones which undergo a reaction (e.g., the generationof EGA, resulting in deprotection). In other words, fluid levelreduction can be performed only in wells where one or more reactants aregenerated.

The construction of the wells can be accomplished by conventionalmanufacturing methods, including, for example, CMOS and VLSI techniques.The wells can be formed in semiconductor or polymeric substrates. In anexemplary embodiment, the wells are configured in a semiconductorsubstrate using conventional etching and boring techniques. The insidersurface of the wells may be coated with insulating material to reducecross talk between adjacent wells. In corollary embodiment, wellsurfaces may be coated to increase conductivity thereby generating EGAmore uniformly. Well surfaces may be coated with different layers toreduce cross-talk while increasing electro- or thermal-conductivityinside the well. Thus, the walls may comprise a composite of differentmaterial which while reducing cross-talk between the wells, wouldincrease conductivity within each well for rapid EGA generation.

The top surface of the wells (the span between adjacent wells) may alsobe coated to provide reagent repellent surfaces. By way of example, thetop surfaces may be coated with hydrophobic compositions to repelcross-contamination. Methods for reducing well to wellcross-contamination are set out in U.S. Pat. No. 6,444,111, thedisclosure of which is incorporated herein by reference.

Finally, the shape of the wells may be configured to reducecross-contamination while increasing reaction speed. For example, thewells may be configured to have cylindrical, barrel or conical shapes.

In many methods using, for example, the plate configuration of FIGS.2A-2B, the sugar group of the first nucleotide is deprotected byactivating (energizing) a chemical reaction initiated by an electricalsignal (or a pulse). As noted elsewhere herein, one method for doingthis is through the generation of an electrochemically generated acid(EGA). In many cases, it will be desirable to control the amount ofchemical reactant made (e.g., EGA) so as to efficiently catalyze thedeprotection reaction while limiting the possibility of reactant fromcross-contamination.

FIG. 17 shows a top view of a channel chip design having threeelectrodes. Counter electrode elements 1700 and 1702 are located at thetop of the two side channels and across the bottom of the flow channel1701. Reference electrodes 1703 surround the two wells with workingelectrodes 1704 are also present.

In order to limit the flow of protons a series of steps may be taken,including (1) the use of buffers which prevent significant pH shifts inthe presence of small amounts of protons, (2) the use of a quinone redoxsystem, and (3) designing the dimensions of wells and channels tomaintain substantial distances between them (e.g., using well volume 150times smaller than according channel volume).

For example, using the schematic shown in FIG. 17 for purposes ofillustration, the distances between the working electrode 1704 andcounter electrode elements 1700 and 1702 may be about 200 μm. Further,interception of protons by base molecules may be used to decrease thenumber of protons that reach other wells. Also, reference electrodestrips 1703 between wells having the same potential as the counterelectrode elements 1700 and 1702 can be used to generate base moleculesand further could prevent proton “cross-talk”. Methods and componentssuch as these, in addition to other methods set out herein, provide forhigh fidelity nucleic acid molecule synthesis.

For purposes of illustration, a prederivatized bead (e.g., a magneticbead) may be placed in wells 1 through 5 of FIGS. 2A-2B with an “A” beadin wells 1 and 5, a “C’ bead in well 2, a “U bead in well 3, and a “G”bead in well 4. All five wells may then be filled with an EGA reagent(e.g., a reagent containing methanol, acetonitrile, hydroquinone,anthraquinone, tetraethylammonium p-toluene sulfonate, and2,6-lutidine). The next base to be added to chain is G and the onlynucleic acid molecule of the molecules to be generated which contains aG at position 2 is in Well 1. Thus, current is applied only to Well 1.This current creates an acidic microenvironment which results indeprotection of the 5′ position of the nucleotide only in Well 1. Aftera fixed (or variable) reaction time, all five wells are washed. Anucleotide, which has the phosphorus, sugar and a base (a T in thisinstance), is added to all of the wells in the presence of a catalyst(e.g., a tetrazole catalyst). After a predefined reaction time, all fivewells are washed and unreacted first nucleotide may be capped to avoidaccumulation of deletions, using, for example, acetic anhydride andN-methylimidazole. Again, after a predetermined reaction time, all fivewells are washed and phosphite triesters formed by chemical reaction maybe oxidized to form the more stable phosphate triester, using, forexample, iodine containing reagents. This process is then repeated untilthe final base of the nucleic acid molecule has been added. Later, thesynthesized nucleic acid molecules may be cleaved from the solidsupport. This may be done, for example, using aqueous or gaseous ammoniawith heating. The cleavage method may vary, however, with factors suchas the linker used.

The amount of current applied to each well and its duration will varywith parameters such as the amount of reagent to be generated and thesize of the well. The applied current may be a pulse of varying shapeand/magnitude. The pulse may define a series of varying amplitude pulses(frequency) or a gradual increase/decrease amplitude. The amplitude andduration of the pulse can be adjusted for the optimum generation ofreagent. As an example, the current applied to a well may be adjustedfor a specified period of time to generate a specified quantity of EGA.The amount of EGA intended for generation will typically be at leastenough sufficient to fully catalyze deprotection of the nucleic acidmolecules present.

In some aspects of the invention, “electrowetting” may be employed. Twoaspect of the invention where electrowetting may be particularly usefulis for the mixing of reagents for (1) nucleic acid synthesis and pooling(Modules 1 and 2) and (2) assembly (Module 3).

In brief, electrowetting involves modifying the surface tension ofliquids on a solid surface using a voltage. Application of an electricfield (e.g., alternating or direct), the contact angle between the fluidand surfaces can be modified. For example, by applying a voltage, thewetting properties of a hydrophobic surface can become increasinglyhydrophilic and therefore wettable. Electrowetting principle is based onmanipulating droplets on a surface comprising an array of electrodes andusing voltage to change the interfacial tension. In some embodiments,the array of electrode is not in direct contact with the fluid. Inadditional embodiments, the array of electrode may be configured such asthe support has a hydrophilic side and a hydrophobic side. The dropletssubjected to the voltage will move towards the hydrophilic side. In someembodiments, the array or pattern of electrodes may be a high densitypattern. When used in conjunction with the phosphoramidite chemistry (aswell as other reagents), the array of electrodes should be able to movedroplets volumes ranging from 1 pL (and less) to 10 pL. Accordingly,aspects of the invention relate to high voltage complementarysemi-conductor microfluidic controller. In some embodiments, the highvoltage complementary semi-conductor device (HV-CMOS) has an integratedcircuit with high density electrode pattern and high voltageelectronics. In some embodiments, the voltage applied is between 15V and30V. Electrowetting methods are set out in U.S. Patent Publication No.2012/0220497 A1, the disclosure of which is incorporated herein byreference.

Electrowetting works by using an electric voltage to alter the shape ofa liquid drop. In some instances, electrowetting involves a sessile droppositioned on a dielectric-coated electrode. When current is applied,the drop flattens and flows out to the sides, thereby wetting additionalsurface. When current is removed, the drop returns to its original shapeand retracts from the areas covered upon current application.Electrowetting are set out in the paper at the following URL:http://www.ll.mit.edu/publications/journal/pdf/vol17_no2/17_2_4Berry.pdf

In some embodiments of the invention, nucleic acid synthesis site mayhave adjacent to is a series of reagents that flow into and recede fromthe synthesis site when current is applied to the correct reagentlocation. Thus, the invention includes methods for the synthesis ofnucleic acid molecules by the addition and removal of reagents from asynthesis site induced by the addition and removal of current fromadjacent reagents. In some instances, the number of reagents adjacent toa nucleic acid synthesis site may be from about 2 to about 10, fromabout 3 to about 10, from about 4 to about 10, from about 5 to about 10,from about 6 to about 10, etc.

Electrowetting methods may also be used for fragment assembly and errorcorrection (Module 3). Thus, the invention includes methods for mixingreagents using electrowetting for the assembly and error correction ofnucleic acid molecules. Reagents that may be contacted with nucleic acidmolecules in these aspects of the invention include exonucleases,mist-match repair endonucleases (MMEs), ligases, buffers, EDTAsolutions, etc.

One problem with electrowetting methods is “splash over” which may occurbetween mixing areas and also because, in many instances, planar orsemi-planar surfaces are used. Thus, unless microfluidic drainagechannels, or the like, are employed, there is a possibility of splashover contamination of mixing areas during reagent changes.

Two means for minimizing this mixing is through the use of microfluidicchannels and barriers. Barrier may be placed (e.g., physical barrierssuch as raised areas) to prevent reagents from moving from one mixingarea to another. After a desired reaction is finished, the barrier maybe removed. Different reactions may be performed sequentially atdifferent and/or overlapping subsets of mixing areas.

As mentioned above, the methods of nucleic acid synthesis may beimplemented and controlled in a system according to various embodimentsdescribed herein by a processor or computing system, such as theexemplary computing system depicted in FIG. 15. For example, applyingcurrent (pulse or continuous wave) to selected wells to generate aspecific quantity of EGA to fully catalyze deprotection may becontrolled by a computing system executing processor executableinstructions according to various embodiments of the present teachings.

Deblocking may also occur through the use of redox systems. Examples ofsuch system systems include hydroquinone/anthraquinone; pH buffer suchas 2,6-lutidine to reduce proton cross talk between active wells andinactive neighboring wells.

Efficient production of nucleic acid molecules may require that nucleicacid synthesis steps be tailored to the molecules being constructed.Consider the example of the construction of nucleic acid moleculesdesigned for construction of viral genome with a CG/AT ratio of 60/40.Nucleic acid molecule building blocks of such a genome will invariablehave more Cs and Gs than As and Ts. In such an instance, it may bedesirable to have more reactions which add Cs and Gs than As and Ts. Asan example, the sequence of base addition may be a repetition of A T C GC A T G C G (SEQ ID NO: 1). Thus, the invention further includeschemical synthesis processes which are tailored for efficient productionof specified nucleic acid molecules. In one aspect, this entails addingbases to nucleic acid molecules during chemical synthesis in mannerwhich reflects or closely approximates the prevalence of the bases inthose molecules.

The invention includes, for example, methods which result in highfidelity, microscale production of nucleic acid molecules. Thus, theinvention includes methods by which nucleic acid molecules are producedwith the following parameters: between 1×10⁵ and 1.5×10⁹ copies of anucleic acid molecule are generated with an average number of basemis-incorporations of between 1 base in 100 and 1 base in 500. Theinvention includes similar methods with the parameters set out in Table3.

TABLE 3 Nucleic Acid Molecule Copies No. of Base Mis-Incorporations(Avg.) 1 × 10⁶ and 1.5 × 10⁹ 1 in 150 to 1 in 500 1 × 10⁶ and 1.5 × 10⁹1 in 150 to 1 in 400 1 × 10⁶ and 1.5 × 10⁹ 1 in 100 to 1 in 300 1 × 10⁶and 1.5 × 10⁹ 1 in 200 to 1 in 400 1 × 10⁶ and 1.5 × 10⁹ 1 in 300 to 1in 1,000 1 × 10⁶ and 1.5 × 10⁹ 1 in 300 to 1 in 2,000 1 × 10⁶ and 1.5 ×10⁹ 1 in 500 to 1 in 4,000 1 × 10⁷ and 1.5 × 10⁹ 1 in 150 to 1 in 500 1× 10⁷ and 1.5 × 10⁹ 1 in 150 to 1 in 400 1 × 10⁷ and 1.5 × 10⁹ 1 in 100to 1 in 300 1 × 10⁷ and 1.5 × 10⁹ 1 in 200 to 1 in 400 1 × 10⁷ and 1.5 ×10⁹ 1 in 300 to 1 in 1,000 1 × 10⁷ and 1.5 × 10⁹ 1 in 300 to 1 in 2,0001 × 10⁷ and 1.5 × 10⁹ 1 in 500 to 1 in 4,000 1 × 10⁷ and 1.5 × 10¹⁰ 1 in150 to 1 in 400 1 × 10⁷ and 1.5 × 10¹⁰ 1 in 100 to 1 in 300 1 × 10⁸ and1.5 × 10¹⁰ 1 in 150 to 1 in 400 1 × 10⁸ and 1.5 × 10¹⁰ 1 in 100 to 1 in300 1 × 10⁸ and 1.5 × 10¹⁰ 1 in 200 to 1 in 400 1 × 10⁸ and 1.5 × 10¹⁰ 1in 300 to 1 in 1,000 1 × 10⁸ and 1.5 × 10¹⁰ 1 in 300 to 1 in 2,000 1 ×10⁸ and 1.5 × 10¹⁰ 1 in 500 to 1 in 4,000

Nucleic acid molecules prepared and used in accordance with theinvention may contain modified nucleic acid molecules including lockednucleic acids (LNA), peptide nucleic acids (PNA), and the like. A PNA isa polyamide type of DNA analog, and the monomeric units for A, G, T, U,and C are available commercially. Furthermore, nucleic acid molecules ofthe invention may comprise one or more modified bases selected from thegroup including, but not limited to, 5-fluorouracil, 5-bromouracil,5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine,5-(carboxyhydroxylmethyl)uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 8-azaguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid,wybutoxosine, pseudouracil, queosine, inosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, 5-methyl-2-thiouracil,3-(3-amino-3-N-2-carboxypropyl)uracil, and 2,6-diaminopurine. The lattermodified base can form three hydrogen bonds when base-paired with dT andcan increase the T^(m) of short nucleic acid molecules by as much as1-2° C. per insertion. This effect, however, is complex and is dependenton sequence context.

2-Aminopurine can substitute for dA in a nucleic acid molecule. It is anaturally fluorescent base that is sensitive to the local environmentmaking it a useful probe for monitoring the structure and dynamics ofDNA hairpins and for detecting the base stacking state of a duplex.2-aminopurine can be destabilizing and slightly lower the T^(m).5-Bromo-deoxyuridine is a photoreactive halogenated base that can beincorporated into nucleic acid molecules to crosslink them to DNA, RNAor proteins with exposure to UV light. Other modified bases such asinverted dT may be incorporated at the 3′-end of a nucleic acidmolecule, leading to a 3′-3′ linkage which inhibits both degradation by3′ exonucleases and extension by DNA polymerases. In another embodimentof the invention an inverted dideoxy-T may be placed at the 5′ end of anucleic acid molecule to prevent unwanted 5′ ligations. A dideoxy-C(ddC) 3′ chain terminator may be used to prevent 3′ extension by DNApolymerases. 5-Methyl deoxy-C when substituted for dC will increase theT^(m) by as much as 0.5° C. per insertion. In one embodiment thenaturally occurring base deoxy-Inosine may be used which is lessdestabilizing than mismatches involving the four standard bases. Thus,the invention provides, in part, compositions and methods relating tothe synthesis of modified nucleic acid molecules with novel propertiesand/or functions.

One modification of the plate format shown in FIG. 2 is to use a “liquidcover” to the wells. One way this could be performed is for the wells tocontain a bilayer. For example, the bottom portion of the wellcontaining the solid support could contain an EGA. Above this could be alower density, optionally non-miscible, fluid. The lower density fluidlayer will prevent or retard the diffusion of acid out of the desiredwell and over to an undesired well. Further, the lower density fluid canbe positioned to make conductive contact with an upper electrode. Oneexample of a commercially available “liquid coverslip” is sold byVentana Medical Systems, Inc (cat. no. 650-010). This product is asolution used as a barrier between the aqueous reagents and the air,which prevents evaporation, and is designed to provide a stable aqueousenvironment for applications such as immunohistochemistry and in situhybridization reactions.

One exemplary protocol for practicing methods of the invention is asfollows. Porous silane-coated magnetic beads (MyOne Beads, Dynal) with auniform diameter of 1 micron are added to the chip surface by controlledor pulsed flow to ensure uniform distribution of the beads across themicrowells (about 1.3 μm diameter) on the chip and to ensure that amaximum number of wells are loaded with one bead. Wells not containing abead are identified by a pre-synthesis current check that delineate theresistance difference among empty wells and well that contain aconductive magnetic bead.

A variety of chemistries are possible in the preparation of the beadsurface. For example, a number of layers of silane can be produced toimpart greater functional surface area to the beads. The silanecoating(s) is/are prepared so that there is stable attachment of thehydroxyl functional group; typically through a trimethoxy or triethoxysilane linker, of the silane core to the naked silica bead surface toexpose a primary hydroxyl group through with the initial amiditesynthetic step is coupled. The fundamental chemistry, developed for aplanar array surface electrode, for initiation and coupling in DNAsynthesis can be found in Maurer et al., Electrochemically GeneratedAcid and Its Containment to 100 Micron Reaction Areas for the Productionof DNA Microarrays, PLoS ONE 1(1): e34. doi:10.1371/journal.pone.0000034(2006).

Fabrication of the chip: Electrode materials such, as iridium metal upto 50 nm thick are produced on oxidized high-resistivity siliconselected for high conductivity and chemical stability under synthesis,reagent addition and deblocking conditions. Electrodes are connected byultrasonic bonding to a printed circuit board to provide digitallycontrolled analogue integrated switch circuits activating electrodeschosen for deblocking a given well. Printed circuit boards are carefullyaligned and bonded to the regular microwell structure to generate thesynthesis chip. A cover plate providing and sealing the interior volumefor reagents and a general complementary circuit electrode is bonded atthe perimeter and over the upper surface of the microwell structure tocomplete the closed synthesis chip.

Conventional semiconductor or polymer material may be used for formingwells 200. For example, CMOS technology can be used to form wells ofdesired shape or size in the semiconductor material such as SiO or SiO₂.Depending on the desired application, electrodes 202 can be fabricateswith wells 200 or separately.

Administration of nucleic acid synthesis (e.g., DNA synthesis) reagentsto the chip can be performed by any number of means. For example, oncethe beads are loaded into the chip, a computing system controls a seriesof reagent additions and washings may be carried out to affectphosphoramidite DNA synthesis on the surface of the beads residing inthe microwells of the chip. Processor-executable instructions may beemployed which determine, for any given population of DNA sequences, theoptimal order of DNA synthesis reagent additions and sequence of reagentadditions and washing steps relative to volume/cost of reagents and timeof a synthesis run. Furthermore, as mentioned above, controller orprocessor-controlled current to specific wells on the chip determine inwhich wells electrochemically generated acid may be produced anddeprotection to activate the growing nucleic acid molecule on the beadin the well may be chemically prepared to couple the next amidite baseadded into the reaction vessel. A number of specific configurations ofapparatus and components for administration of synthesis reagents and toensure precise and controlled fluid administration are possible throughan optimized development process. Phosphoramidite DNA synthesis steps,conditions and reagents using EGA to affect deprotection can be foundin, for examples, Maurer et al., Electrochemically Generated Acid andIts Containment to 100 Micron Reaction Areas for the Production of DNAMicroarrays, PLoS ONE 1(1): e34. doi:10.1371/journal.pone.0000034 (2006)and Egeland and Southern, Electrochemically directed synthesis ofoligonucleotides for DNA microarray fabrication, Nucleic Acids Research,33(14):e125 (2005).

Composition and concentration of EGA components: The exact compositionand concentration of EGA reagent is influenced by the preciseconductive, structural and geometric properties of the electrodes andmicrowells and the parameters associated with the application (current,voltage and time) of current to convert the EGA to its acid forms.Generally, the smaller the volume for EGA production to affectdeprotection, the smaller the required current strength and/or time ofcurrent application. Since the amount of nucleic acid molecule producedin such microscale systems falls below a threshold that can be directlyand accurately measured, surrogate assays, such as hybridization orproduct enrichment following target amplification, for nucleic acidmolecule synthesis and coupling efficiency are typically required. EGAreagents, including hydroxyquinone and benzoquinone, withtetrabutylammonium hexafluorophosphate in anhydrous acetonitrile areused to generate electrochemical acid via anodic oxidation to affectdeprotection. EGA reagents above in concentration up to 25 mM areprepared and administered to the chip prior to the application ofcurrent to affect deprotection. In determination of the optimalparameters, it will generally be desirable to avoid base damage causedby depurination from over-exposure of DNA to acid.

Application of current to affect EGA-based DMT deprotection: Current maybe applied constantly up to 2 μA and voltage up to 2 V is applied to anelectrode in the controlled circuit for a time period of up to 30seconds. Current may also be applied in pulse durations from 10 to 2000ms during a time of 1 to 60 seconds. Current may also be applied as invarious pulses (e.g., from about two to about 10,000, from about ten toabout 10,000, from about fifty to about 10,000, from about 100 to about10,000, from about 1,000 to about 10,000, from about ten to about 500,etc. pulses) up to 2 μA (e.g., from about 0.02 nA to about 20,000 nA,from about 0.2 nA to about 20,000 nA, from about 0.2 nA to about 5,000nA, from about 0.2 nA to about 2,000 nA, from about 0.2 nA to about1,000 nA, from about 0.2 nA to about 5000 nA, from about 2.0 nA to about20,000 nA, from about 2.0 nA to about 10,000 nA, from about 2.0 nA toabout 5,000 nA, from about 2.0 nA to about 2,000 nA, from about 5.0 nAto about 20,000 nA, from about 5.0 nA to about 8,000 nA, from about 10nA to about 20,000 nA, from about 10 nA to about 8,000 nA, from about 10nA to about 5,000 nA, from about 20 nA to about 20,000 nA, from about 20nA to about 8,000 nA, from about 50 nA to about 20,000 nA, from about 50nA to about 10,000 nA, from about 50 nA to about 5,000 nA, from about100 nA to about 10,000 nA, from about 500 nA to about 20,000 nA, fromabout 500 nA to about 10,000 nA, from about 500 nA to about 5,000 nA,from about 1,000 nA to about 20,000 nA, from about 1,000 nA to about10,000 nA, etc.). In some instances, current will be pulsed for anywherefrom about 1 second to about 30 seconds, from about 2 second to about 30seconds, from about 4 second to about 30 seconds, from about 5 second toabout 30 seconds, from about 5 second to about 20 seconds, from about 5second to about 15 seconds, from about 5 second to about 10 seconds,etc. Of course, efficient deprotection and nucleic acid moleculesynthesis must be determined as the exact composition and concentrationof EGA reagent is influenced by the precise conductive, structural andgeometric properties of the electrodes and microwells and the parametersassociated with the application (current, voltage and time) of current.

In certain embodiments of the invention the nucleic acid molecule or aportion thereof may be subject to a sequence optimization process priorto synthesis. Different computational approaches for sequencemodification are known in the art and may be employed to optimize agiven nucleotide sequence in terms of 1) efficient assembly and/or 2)improved performance in a given host. To design a nucleotide sequencefor optimal assembly, a full-length sequence may be broken down into adefined number of smaller fragments with optimal hybridizationproperties by means of an algorithm taking into account parameters suchas melting temperature, overlap regions, self-hybridization, absence orpresence of cloning sites and the like. In certain aspects of theinvention, at least part of the desired nucleic acid sequence may encodea polypeptide or protein. In such cases, it may be desirable to optimizethe open reading frame for improved performance in a given homologous orheterologous host, such as expression yield or solubility. An increasein gene expression may be achieved, for example, by replacingnon-preferred or less preferred codons by preferred codons or byincreasing the number of CpG dinucleotides in the open reading frame asdescribed, for example, in U.S. Pat. Nos. 5,786,464 and 6,114,148 andU.S. Patent Publication No. 2009/0324546 AA, the disclosures of whichare incorporated herein by reference.

In one specific embodiment, an optimized open reading frame may becombined with an algorithm to encrypt a secret message into the openreading frame as described in U.S. Patent Publication No. 2011/0119778AA. Such message may allow the identification or tracking of certainsynthetic nucleic acid molecules. In certain aspects of the invention,it may be desired to use an optimization strategy that takes intoaccount multiple different parameters simultaneously includingassembly—as well as expression-related sequence properties. One exampleof a comprehensive multiparameter approach that may be used in thecurrent invention for optimized sequence design is the GENEOPTIMIZER®technology described in U.S. Patent Publication No. 2007/0141557 AA, thedisclosure of which is incorporated herein by reference. Thus, theinvention provides in part aspects of optimal sequence design fordownstream applications including assembly and expression strategies.

Module 2

After completion of a synthesis run on Module 1, support-associated(e.g., bead-associated) nucleic acid molecules may be subject topost-processing in Module 2. Processes performed in Module 2 may beperformed manually or by computer directed automation controlling suchsteps as picking and pooling of a bead (e.g., a magnetic bead) from thesynthesis microwell array and vapor-phase cleavage and deprotection toprepare the nucleic acid molecules for subsequent assembly steps, asappropriate.

To expose a microwell array of bead-attached nucleic acid molecules, thecover of the synthesis well, when present, may be removed. In oneembodiment, the cover is removed by automatic means in acomputer-controlled manner.

A bead picking instrument comprising, for example, aprecision-controlled electro-micromagnet can be programmed andcontrolled to extract and pool individual beads harboring synthesizednucleic acid molecules. Depending on the application and the number ofnucleic acid molecules to be assembled, all of the beads of themicrowell array may be pooled or only a subset of the beads. When only asubset of the beads are pooled or when the total number of beads islimited, the number of beads pooled may vary widely and include fromabout 10 to about 50, from about 50 to about 100, from about 100 toabout 1000, from about 50 to about 10,000, from about 100 to about10,000, or from about 500 to about 10,000 individual beads. These beadsmay be deposited in any suitable container. One example of a containeris the well of a microwell plate (e.g., a well of a 1536 microwellplate).

Automation suitable use with the invention includes aprecision-controlled electromicromagnet picks up the first bead anddeposits it into a pooling well (i.e., a well which contains multiplebeads for collection of nucleic acid molecules sought to be used incombination). Alternatively, a precision-controlled electromicromagnetcan be used which picks up the first bead and then moves in the X-Ydirection to the next position, lowers down in the Z direction to pickup the second bead, back up in the Z direction to get out of themagnetic field range, moves to the third well in the X-Y direction, etc.Thus, the magnet is left “on” and the set of beads (e.g., from about twoto about fifty, from about ten to about fifty, from about two to aboutone hundred, from about ten to about one hundred, from about twenty toabout eighty, etc.) is picked up and carried as a string of beads. As aset of beads is collected, this set is then deposited in simultaneouslydeposited into a pooling well. Of course, multiple sets of beads may becollected and deposited in a single pooling well.

In some instances, beads may be extracted and pooled using systems asdescribed, for example, in U.S. Patent Publication Nos. 2008/0281466 AAor 2008/0113361 AA or in U.S. Pat. Nos. 6,887,431; 7,347,975 or7,384,606, the disclosures of which are incorporated herein byreference. In other embodiments of the invention a bead pickinginstrument with at least one integrated precision-controlledelectro-micromagnet may be used. Such a picking instrument may becontrolled by a control unit which can be programmed to control themovement of the micromagnet to align with specific microwells. In afurther embodiment, the control unit may provide means to control theadjustment of the distance between the micromagnet and the microwell. Ina specific embodiment, the micromagnet may be controlled and activatedby electric means to allow extraction of single magnetic beads carryinga specific nucleic acid sequence.

Electro-micromagnets used in the current invention may be hollow magnetsor needle shaped and will often be of a size and dimension to focus themagnetic field at its tip to allow for specific targeting of individualbeads. In a specific embodiment, the micromagnet may be composed of anelectro-magnet and a permanent magnet wherein the activity of thepermanent magnet can be controlled by the electro-magnet.Electro-micromagnet used in conjunction with the invention may be in anynumber or format and may, for example, comprise a single magnet or bearranged together with other micromagnets in a row.

In certain embodiments of the invention, an electro-micromagnet may beused to extract and pool all magnetic beads contained in the microwellsof a single arrays. For this purpose, the electro-micromagnet may beallocated to each microwell to extract the bead-attached nucleic acidmolecules in a step-wise manner in a pre-defined or random order. In oneembodiment, all nucleic acid molecules required for the assembly of afull-length construct may be synthesized on a single array. According tothe amount of nucleic acid molecules required to build a full-lengthconstruct, arrays of different sizes and dimensions can be used.

In another embodiment, the electro-micromagnet may be programmed totarget only a portion of the microwells of a specific array to extractand pool a predefined selection of bead-attached nucleic acid molecules.The electro-micromagnet can be programmed to extract and pool beads fromthe microwells of two or more different plates. The picking may combinefull extraction of all beads of a first plate with selective extractionof a portion of beads obtained from a second plate. The first and thesecond plate may vary in size and dimension.

Each magnetic bead extracted by the micromagnet may then be transferredto a pooling station by moveable means of the picking instrument. In oneembodiment the pooling station may contain a chamber with a microwellplate. In one embodiment the microwell plate may be a 1536 microwellplate. However, microwell plates of other sizes and dimensions (e.g.,standard 96 well plates) are known in the art and can be used in thecurrent invention. Defined fractions of nucleic acid molecules can bepooled in individual wells of a microwell plate wherein one pooledfraction contains all nucleic acid molecules required to assemble atleast a defined fragment of a full-length construct. In one embodiment,an individual nucleic acid molecule pool may contain all nucleic acidmolecules required to assemble a full-length construct. Differentnucleic acid molecule pools allocated to each well can be furtheridentified using a machine readable identifier disposed on the microwellplates.

Electrostatic forces may also be used to remove beads and othersubstrates from synthesis platforms. Using FIGS. 2A-2B for purposes ofillustration, oligonucleotide synthesis substrates (beads in thisinstance) may have an electrostatic charged and separated fromassociation with a surface or well using an opposite charge. Forexample, if one or more beads shown in FIGS. 2A-2B have a positivecharge then the lower electrode may be used to generate a positivecharge to repel the bead and force it from the well. Magnetic chargescan also be used to achieve the same purpose. Residual magnetism mayalso be employed. In essence, residual magnetism is magnetism thatremains in a material after being exposed to magnetic force. In manyinstances, magnetic substrates will be of small size. Thus, attractionof such substrates will typically not requires strong magnetic fields.Residual magnetism may be present in the substrate a selection probeused to bind to the substrate or both. Further, charges may be used toselectively remove a subset of synthesis substrates from a synthesisplatform.

Electrostatic forces for required for the removal of beads and othersubstrates from synthesis platforms can be readily calculated. Table 4below assumes a relative homogeneous electrical field is present andthat each bead acts as a single charge point. Nucleic acid moleculescarry with them a charge which should be taken into consideration whencharge is used to extrude a bead from a well. Further, charge need onlybe applied to wells that contain substrates with desired nucleic acidmolecules (e.g., nucleic acid molecules for assembly into larger nucleicacid molecules.

TABLE 4 Number of Charge per Electrode Strands Strand (As) Voltage (V)Electrode Distance (m) 1,000,000 1.6 × 10⁻¹⁹ 2 1.00 × 10⁻⁵ ElectrodeDistance (μm) 10 Point Electric Field Charge (As) Strength (V/m) Force N1.6 × 10⁻¹³ 200000 0.000000032 Electric Field Strength (V/mm) Force μN200 0.032

In another embodiment, a synthesis platform may contain a series ofregions that separate from other regions of the synthesis platform. Forexample, a synthesis platform may contain 100 rows of synthesis areas ina square 10×10 arrangement. Further, the synthesis platform may bedesigned so that it is separatable into ten rows of ten synthesis areas.For purposes of illustration, assume that one seeks to produce eightdifferent assembled nucleic acid molecules and these assembled nucleicacid molecules are designed to be formed from the assembly of thefollowing number or oligonucleotides:

TABLE 5 No. of Row Oligos No. Assembled Nucleic No. 1 7 1 2 8 2 3 8 3 49 4 Assembled Molecule No. 5 9 5 6 10 6 7 13 7-8  8 15 9-10

Table 5 indicates the numerical designation of the various assemblednucleic acid molecules, the number of oligonucleotides that will be usedto assemble the assembled nucleic acid molecules, and the rows in whichthe oligonucleotides are synthesized in. In this embodiment, rows 1-5will each have at least one synthesis area in which no oligonucleotideswill be produced.

After synthesis is completed, the separatable rows may be separated andthe synthesized nucleic acid molecules, collected/processed andassembled, for example, as described elsewhere herein.

Other methods may also be used to collect nucleic acid synthesissubstrates, including (1) “grabbing”, for example by the use of tweezerslike devices which operate based upon mechanical (e.g., actualgrabbing), optical, sonic, magnetic principles, (2) “destroying”structures surrounding nucleic acid synthesis substrates by methods suchas chemical dissolution or through the use of lasers, (3) moving nucleicacid synthesis substrates by, for example, the use of thermal,electrostatic, magnetic, fluidic energy, (4) hybrid gripper whichcombine, for examples, (a) magnetic and fluidic flushing, (b) magneticand piezoelectric methods, and (c) electrostatic lifting and fluidicflushing, (5) magnetic fixing/collecting using, for example, modulatedpermanent magnets, external coils, planar coils on synthesis substrates,etc., (6) electrostatic lifting & collecting, and (7) flux direction(e.g., the addition of fluid to the bottom of a well to liftsubstrates).

Pooling stations used in the practice of the invention may furthercontain a microwell handling device which comprises controllablemoveable means for moving the microwell plate from a first to at least asecond position in X and/or Y and/or Z direction and can be programmedto perform liquid handling steps. Such pooling stations may further beequipped with a pipetting device and a suction apparatus allowing forcontrolled addition and removal of reagents. Alternatively the removalof liquid can be performed by vacuum means. The pipetting device mayfurther be connected to reagent reservoirs and mixing means to mix andadd defined amounts of reagents required for purification and subsequentprocessing and assembly steps. Integrated liquid handling devicescombining the respective functions are known by those skilled in theart.

In a specific embodiment, the pooling station integrates means to allowfor further combining of one or more nucleic acid molecule pools fromfirst and second wells into a third well to yield a larger nucleic acidmolecule pool. Such step-wise pooling may be required in cases wherevariants or libraries of full-length constructs are assembled fromidentical and variable sequence elements.

Pooling stations used in the practice of the invention may furthercontain a magnet located beneath the microwell plate. In a specificembodiment such a plate magnet may serve as counterpart to themicromagnet in order to trigger release of the extracted beads into therecipient microwell. Alternatively the electro-micromagnet may be ahollow magnet connected to a capillary that can be flushed with liquidto blow out the bound bead into the recipient well. Other means of beadrelease may also be employed.

With respect to pooling of nucleic acid molecules, this may be done anynumber of ways. For example, synthesis substrates may be collected andplaced in a single contained. Alternatively, nucleic acid molecules maybe released from synthesis substrates and then contacted with eachother. Further, nucleic acid molecules may be assembled byhybridization. This means that more than one assembly may occur in thesame container. In other words, the invention includes methods by whichassembly of more than one (e.g., two, three, four, five, six, etc.)nucleic acid molecule occurs from smaller, chemically synthesizednucleic acid molecules. One application where the assembly of more thanone larger nucleic acid molecule (e.g., replicable nucleic acidmolecules) may be useful is where the assembled nucleic acid moleculesare intended for insertion into the same cell. Thus, one of theassembled nucleic acid molecules could be a chromosome and another couldbe a plasmid.

Once desired pools of nucleic acid molecules have been generated,bead-attached nucleic acid molecules will often be further processed,for example, to obtain functional nucleic acid molecules for downstreamreactions. After chain synthesis the 5′-terminal 5′-hydroxy group isusually protected, for example, with a dimethoxytrityl (DMT) group; theinternucleosidic phosphate or phosphorothioate moieties may also beprotected, for example, with 2-cyanoethyl groups; and the exocyclicamino groups in all nucleic bases (except for T and U) may be protected,for example, with acyl protecting groups. Usually, the 5′-terminal DMTgroup is cleaved after the last synthesis cycle on the support beforethe bead-attached nucleic acid molecules are pooled. However, allprotection groups have to be removed in a deprotection step before thenucleic acid molecules can be effectively used in subsequent processes.

In one embodiment of the invention, deprotection is performed, forexample, without releasing the nucleic acid molecule form the bead. Thiscan be carried out by choosing a base-stable, non-cleavable linker.Respective linkers are known by the skilled person.

In one embodiment, nucleic acid molecules are released from the beadsprior to downstream assembly. If cleavage of nucleic acid molecule isrequired, cleavage and deprotection may be performed in a single step.Release of the nucleic acid molecules may be achieved by cleaving thelinker attaching the 3′-end of the nucleic acid molecule to the bead(e.g., a magnetic bead) with a suitable reagent. Suitable reagents andconditions for cleavage depend on the nature of the linkage as describedelsewhere herein and are known by those skilled in the art.

In one embodiment of the invention, nucleic acid molecules are attachedto the magnetic beads via succinyl groups. The succinyl linker may becleaved by the use of, for example, concentrated aqueous ammoniumhydroxide. The reaction is usually carried out at temperatures between50° C. and 80° C. for at least one to about eight hours. Of course,cleavage conditions may vary depending on the protocol and theprotecting groups used. The ammonia solution may then removed byevaporation, leaving the nucleic acid molecules ready for purification.

In one embodiment, cleavage may be carried out by vapor-phaseprocessing. In vapor-phase processing, nucleic acid molecules may becleaved in a closed chamber in a gaseous environment comprising gaseouscleavage/deprotection reagent, such as gaseous ammonia or ammoniumhydroxide vapors. Respective methods are set out, for example, in U.S.Pat. Nos. 5,514,789 or 5,738,829, the disclosures of which areincorporated herein by reference.

The above reaction will typically also triggers cleavage of otherprotecting groups including the cyanoethyl group, the group protectingthe heterocyclic primary amine and the DMT group on the very last base.Thus, a single cleavage reaction may be used, when appropriate, toremove all protecting groups present.

Linkers used in the practice of the invention may be cleaved using atleast two approaches: (a) simultaneously under the same conditions asthe deprotection step or (b) subsequently utilizing a differentcondition or reagent for linker cleavage after the completion of thedeprotection step. Various methods to remove universal linkers from anucleic acid molecule are described in the art such as, for example,U.S. Patent Publication No. 2002/0143166 A1, the disclosure of which isincorporated herein by reference.

For downstream applications, it may be required to purify the pooled anddeprotected nucleic acid molecules to remove the cleaved groups, forexample, by precipitation. It may further be required to separate thenucleic acid molecule mixture from the magnetic particles or othersupport. In one embodiment, a plate magnet located beneath the microwellplate can be used to immobilize the beads in the wells while the nucleicacid molecules can be eluted, for example, by suction. Alternatively, inthe absence of a plate magnet, the beads may be automatically removedfrom the wells by magnetic means while the nucleic acid molecules wouldbe retained in the well to obtain femtomoles of individual pools of highquality nucleic acid molecules at picomole concentration ready forfurther processing or use.

In some instances, nucleic acid molecules may be separated from solidsupport while the solid supports remain localized in the same or similarlocation as to where the nucleic acid molecules were synthesized. Insuch instances, typically after synthesis completion, oligonucleotidesynthesis reagents may be removed from contact with synthesis supports,followed by the addition of one or more reagents for release of theconstructed oligonucleotide, also referred to as cleavage reagents.These releasing reagents may be in forms such as liquid or gaseous.Gaseous reagents are referred to above.

In many instances, the cleavage reagent agent will be volatile (e.g., itcan be removed via freeze drying) and non-ionic. The cleavedoligonucleotides may then be recovered by either removal from wells,when present, or by rinsing the synthesis substrate. When microwells areemployed for synthesis, cleavage reagents in liquid form may be used.The synthesis substrate may be coated with such liquid reagents followedby either group removal of synthesized oligonucleotides or removal ofindividual oligonucleotides (less than all of oligonucleotides present).Removal of individual oligonucleotides may be achieved, for example, bylimiting agitation of the substrate and site specific removal (e.g.,with a pipette tip) of fluid containing individual oligonucleotidesafter cleavage has occurred. Such methods will be particularly usefulwhen the substrate contains wells or cavities.

Optionally, synthesized nucleic acid molecules may be concentrated stepafter pooling, cleavage and/or deprotection but to entering into Module3 processes. One concentration method of such concentration would be byan additional second binding, washing, and elution series of sets toreduce the final volume. This increased concentration will increase theconcentration of synthesized nucleic acid molecules, resulting inaccelerated hybridization of overlapping segments in sub-fragmentgeneration as may be desired. Concentration to an increasedconcentration may also be used to “normalize” the concentration ofmultiple pools to a more constant range so that a limited set of, forexample, assembly conditions need be employed in Module 3 processes(e.g., all Module 3 processes).

FIG. 13 shows two methods by which synthesized oligonucleotides may beseparated from supports. In this figure, oligonucleotides have beensynthesized on beads 1300 and released into the surrounding well of amicrowell titer plate 1301. In each instance, wells containingoligonucleotides for collection are covered with fluid 1302 and pipettetips 1303 are used to collect that fluid. On the left side of the figureare two wells where the fluid is contained within the wells. Further tothe right side of FIG. 13, a barrier 1304 extends above the wells toallow fluid to collect at a higher level. In both instances, the fluidmay be there before the pipette tips are brought into close proximity orthe fluid may be delivered by the pipette tips. Also, fluid surroundingthe beads 1300 may be circulated to distribute released oligonucleotidesby flow delivered by the pipette tips 1303.

Module 3

Once the chemical synthesis phase has been completed, the resultingnucleic acid molecules may be assembled, if desired, into larger nucleicacid molecules. Depending on the end purpose for which the final nucleicacid molecules are to be used, the “quality” (e.g., from a sequencefidelity perspective) of the chemically synthesized nucleic acidmolecules may be too low for the intended application. As an example, ifthe chemically synthesized nucleic acid molecules are to be used as longprobes, then they may be of sufficient quality for that purpose withoutfurther processing. However, consider the situation where one hundrednucleic acid segments are to be assembled, each nucleic acid segment isone hundred base pairs in length and there is one error per fifty basepairs. The net result is that there will be, on average, 200 sequenceerrors in each 10,000 base pair assembled nucleic acid molecule. If oneintends, for example, to express one or more proteins from the assemblednucleic acid molecule, then the number of sequence errors would likelybe considered to be too high. Also, while sequencing of individualnucleic acid molecules may be performed, this is time consuming andinvolves additional cost. Thus, in many instances, an error removal stepmay be performed. Typically, this will be performed after a first roundof assembly. Thus, in one aspect, methods of the invention involve thefollowing (in this order or different orders):

1. Fragment Amplification and Assembly (e.g., PCR/in vitro assembly).

2. Error Correction.

3. Final Assembly (e.g., in vivo assembly).

In various embodiments of the present disclosure, error removal stepsmay also be implemented by executing processor-executable instructions.The invention thus includes software based instructions for performingmechanical functions associated with error removal processes, as well asother aspects of the invention.

Any number of methods may be used for fragment amplification andassembly. One exemplary method is described in Yang et al., NucleicAcids Research, 21:1889-1893 (1993) and U.S. Pat. No. 5,580,759, thedisclosure of which is incorporated herein by reference.

In the process described in the Yang et al. paper, a linear vector ismixed with double stranded nucleic acid molecules which share sequencehomology at the termini. An enzyme with exonuclease activity (i.e., T4DNA polymerase, T5 exonuclease, T7 exonuclease, etc.) is added whichpeels back one strand of all termini present in the mixture. The “peeledback” nucleic acid molecules are then annealed incubated with a DNApolymerase and deoxynucleotide triphosphates under condition which allowfor the filling in of single-stranded gaps. Nicks in the resultingnucleic acid molecules may be repaired by introduction of the moleculeinto a cell or by the addition of ligase. Of course, depending on theapplication and work flow, the vector may be omitted. Further, theresulting nucleic acid molecules, or sub-portions thereof, may beamplified by polymerase chain reaction.

Other methods of nucleic acid assembly include those described in U.S.Patent Publication Nos. 2010/0062495 A1; 2007/0292954 A1; 2003/0152984AA; and 2006/0115850 AA and in U.S. Pat. Nos. 6,083,726; 6,110,668;5,624,827; 6,521,427; 5,869,644; and 6,495,318, the disclosures of whichare incorporated herein by reference.

A method for the isothermal assembly of nucleic acid molecules is setout in U.S. Patent Publication No. 2012/0053087, the disclosure of whichis incorporated herein by reference. In one aspect of this method,nucleic acid molecules for assembly are contacted with a thermolabileprotein with exonuclease activity (e.g., T5 polymerase) a thermostablepolymerase, and a thermostable ligase under conditions where theexonuclease activity decreases with time (e.g., 50° C.). The exonuclease“chews back” one strand of the nucleic acid molecules and, if there issequence complementarity, nucleic acid molecule will anneal with eachother. The thermostable polymerase fills in gaps and the thermostableligase seals nicks. Methods like this may be used in conjunction withequipment of FIG. 16. Further, more than one nucleic acid molecule maybe stored with other suitable reagents in the individual storage unitsof 1609 and these storage units may be set to a temperature of, forexample, of 50° C. for assembling the stored molecules.

One commercially available kit which may be used to assemble nucleicacid molecules of the invention, as well as for the insertion of suchnucleic acid molecules into vectors is the GENEART® Seamless Cloning andAssembly Kit (cat. no. A13288), available from Life Technologies Corp.,Carlsbad, Calif.

Single-stranded binding proteins such as T4 gene 32 protein and RecA, aswell as other nucleic acid binding or recombination proteins known inthe art, may be included, for example, to facilitate nucleic acidmolecules annealing.

In some instances, nucleic acid molecules may be amplified on solidsupports. Thus, the invention includes methods where nucleic acidmolecules are synthesized but are not cleaved from solid supports theyare synthesized on. In such instances, the amplified nucleic acidmolecules may be used directed (e.g., as probes) or assembled asdescribed elsewhere herein.

One method for assembling nucleic acid molecules (FIG. 3) involvesstarting with overlapping nucleic acid molecules which are “stitched”together using PCR. In many instances, the stitched nucleic acidmolecules will be chemically synthesized and will be less than 100nucleotides in length (e.g., from about 40 to 100, from about 50 to 100,from about 60 to 100, from about 40 to 90, from about 40 to 80, fromabout 40 to 75, from about 50 to 85, etc. nucleotides). A processsimilar to that shown in FIG. 3 is set out in U.S. Pat. No. 6,472,184,the disclosure of which is incorporated herein by reference. Primers mayalso be used which contain restriction sites for instances whereinsertion into a cloning vector is desired. One suitable cloning systemis referred to as Golden Gate which is set out in various forms in U.SPatent Publication No. 2010/0291633 A1 and PCT Publication WO2010/040531, the disclosures of which are incorporated herein byreference. Thus, where desirable, assembled nucleic acid molecules maybe directly inserted into vectors and host cells. This may beappropriate when the desired construct is fairly small (e.g., less than5 kilobases). Type IIs restriction site mediated assembly may be used toassemble multiple fragments (e.g., two, three, five, eight, ten, etc.)when larger constructs are desired (e.g., 5 to 100 kilobases).

An alternative method for PCR-based assembly of nucleic acid molecules(e.g., chemically synthesized nucleic acid molecules) is based on thedirect ligation of overlapping pairs of 5′-phosphorylated nucleic acidmolecules (“ligation-based assembly”). In this process, single-strandednucleic acid molecules are synthesized, phosphorylated and annealed toform double-stranded molecules with complementary overhangs (e.g.,overhangs of four nucleotides). The individual double stranded moleculesare then ligated to each other to form larger constructs. In certainembodiments this method may be desirable over PCR methods in particularwhere highly repetitive sequences, such as GC stretches are to beassembled. This method may be used to assemble from about two to aboutforty nucleic acid molecules (e.g., from about two to about forty, fromabout three to about forty, from about five to about forty, from abouteight to about forty, from about two to about thirty, from about two toabout twenty, from about two to about ten, etc. nucleic acid molecules).A related method is described in U.S. Pat. No. 4,652,639, the disclosureof which is incorporated herein by reference.

In many instances when ligation-based assembly is employed usingchemically synthesized nucleic acid molecules, the molecules will beless than 100 base pairs in length. Also, the complementary overlaps maybe used for joining the nucleic acid molecules will generally be betweentwo and ten (e.g., from about two to about ten, from about four to aboutten, from about five to about ten, from about two to about eight, fromabout three to about seven, etc. nucleotides in length) (FIG. 4).

One process that may be used to assemble nucleic acid molecules isRed/ET recombination. This process employs E. coli based homologousrecombination mediated by phage protein pairs, such as RecE/RecT orRedα/Redβ. This process is not limited by nucleic acid size and isindependent of restriction sites. Essentially any DNA molecule in E.coli of almost any size can be engineered at any site using Red/ETrecombination. In essence, Red/ET recombination involves threesteps/conditions. The first step or condition is the presence ofhomology arms (e.g., arms of 50 base pairs in length) in linear DNA. Thesecond step or condition is the insertion or presence of the linear DNAin an E. coli cell. The third step or condition is the expression orpresence of any appropriate phage pair (e.g., RecE/RecT or Redα/Redβ) inthe E. coli cell. Red/ET recombination is set out in U.S. Pat. Nos.6,355,412 and 6,509,156, the disclosures of which are incorporatedherein by reference.

Further, as shown in FIG. 4, multiple rounds of polymerase chainreactions may be used to generate successively larger nucleic acidmolecules.

In most instances, regardless of the method by which a larger nucleicacid molecule is generated from chemically synthesized nucleic acidmolecules, errors from the chemical synthesis process will be present.Thus, in many instances, error correction will be desirable. Errorcorrection can be achieved by any number of means. One method is byindividually sequencing chemically synthesized nucleic acid molecules.

Another method of error correction is set out in FIG. 6. FIG. 6 is aflow chart of an exemplary process for synthesis of error-minimizednucleic acid molecules. In the first step, nucleic acid molecules of alength smaller than that of the full-length desired nucleotide sequence(i.e., “nucleic acid molecule fragments” of the full-length desirednucleotide sequence) are obtained. Each nucleic acid molecule isintended to have a desired nucleotide sequence that comprises a part ofthe full length desired nucleotide sequence. Each nucleic acid moleculemay also be intended to have a desired nucleotide sequence thatcomprises an adapter primer for PCR amplification of the nucleic acidmolecule, a tethering sequence for attachment of the nucleic acidmolecule to a DNA microchip, or any other nucleotide sequence determinedby any experimental purpose or other intention. The nucleic acidmolecules may be obtained in any of one or more ways, for example,through synthesis, purchase, etc.

In the optional second step, the nucleic acid molecules are amplified toobtain more of each nucleic acid molecule. The amplification may beaccomplished by any method, for example, by PCR. Introduction ofadditional errors into the nucleotide sequences of any of the nucleicacid molecules may occur during amplification.

In the third step, the amplified nucleic acid molecules are assembledinto a first set of molecules intended to have a desired length, whichmay be the intended full length of the desired nucleotide sequence.Assembly of amplified nucleic acid molecules into full-length moleculesmay be accomplished in any way, for example, by using a PCR-basedmethod.

In the fourth step, the first set of full-length molecules is denatured.Denaturation renders single-stranded molecules from double-strandedmolecules. Denaturation may be accomplished by any means. In someembodiments, denaturation is accomplished by heating the molecules.

In the fifth step, the denatured molecules are annealed. Annealingrenders a second set of full-length, double-stranded molecules fromsingle-stranded molecules. Annealing may be accomplished by any means.In some embodiments, annealing is accomplished by cooling the molecules.

In the sixth step, the second set of full-length molecules are reactedwith one or more endonucleases to yield a third set of moleculesintended to have lengths less than the length of the complete desiredgene sequence. The endonucleases cut one or more of the molecules in thesecond set into shorter molecules. The cuts may be accomplished by anymeans. Cuts at the sites of any nucleotide sequence errors areparticularly desirable, in that assembly of pieces of one or moremolecules that have been cut at error sites offers the possibility ofremoval of the cut errors in the final step of the process. In anexemplary embodiment, the molecules are cut with T7 endonuclease I, E.coli endonuclease V, and Mung Bean endonuclease in the presence ofmanganese. In this embodiment, the endonucleases are intended tointroduce blunt cuts in the molecules at the sites of any sequenceerrors, as well as at random sites where there is no sequence error.

In the last step, the third set of molecules is assembled into a fourthset of molecules, whose length is intended to be the full length of thedesired nucleotide sequence. Because of the late-stage error correctionenabled by the provided method, the set of molecules is expected to havemany fewer nucleotide sequence errors than can be provided by methods inthe prior art.

The process set out above and in FIG. 6 is also set out in U.S. Pat. No.7,704,690, the disclosure of which is incorporated herein by reference.Furthermore, the process described above may be encoded onto acomputer-readable medium as processor-executable instructions.

Another process for effectuating error correction in chemicallysynthesized nucleic acid molecules is by a commercial process referredto as ERRASE™ (Novici Biotech). Error correction methods and reagentsuitable for use in error correction processes are set out in U.S. Pat.Nos. 7,838,210 and 7,833,759, U.S. Patent Publication No. 2008/0145913A1 (mismatch endonucleases), and PCT Publication WO 2011/102802 A1, thedisclosures of which are incorporated herein by reference.

Exemplary mismatch endonucleases include endonuclease VII (encoded bythe T4 gene 49), RES I endonuclease, CEL I endonuclease, and SPendonuclease or methyl-directed endonucleases such as MutH, MutS orMutL. The skilled person will recognize that other methods of errorcorrection may be practiced in certain embodiments of the invention suchas those described, for example, in U.S. Patent Publication Nos.2006/0127920 AA, 2007/0231805 AA, 2010/0216648 A1, 2011/0124049 A1 orU.S. Pat. No. 7,820,412, the disclosures of which are incorporatedherein by reference.

Another schematic of an error correction method is shown in FIG. 7.

Synthetically generate nucleic acid molecules typically have error rateof about 1 base in 300-500 bases). Further, in many instances, greaterthan 80% of errors are single base frameshift deletions and insertions.Also, less than 2% of errors result from the action of polymerases whenhigh fidelity PCR amplification is employed. In many instances, mismatchendonuclease (MME) correction will be performed using fixed protein:DNAratio.

One error correction methods involves the following steps. The firststep is to denature DNA contained in a reaction buffer (e.g., 200 mMTris-HCl (pH 8.3), 250 mM KCl, 100 mM MgCl₂, 5 mM NAD, and 0.1% TRITON®X-100) at 98° C. for 2 minutes, followed by cooling to 4° C. for 5minutes, then warming the solution to 37° C. for 5 minutes, followed bystorage at 4° C. At a later time, T7 endonuclease I and DNA ligase areadded the solution 37° C. for 1 hour. The reaction is stopped by theaddition EDTA. A similar process is set out in Huang et al.,Electrophoresis 33:788-796 (2012).

Another method for removal of error from chemically synthesized nucleicacid molecules is by selection of nucleic acid molecules having correctnucleotide sequences. This may be done by the selection of a singlenucleic acid molecule for amplification, then sequencing of theamplification products to determine if any errors are present. Thus, theinvention also includes selection methods for the reduction of sequenceerrors. Methods for amplifying and sequence verifying nucleic acidmolecules are set out in U.S. Pat. No. 8,173,368, the disclosure ofwhich is incorporated herein by reference. Similar methods are set outin Matzas et al., Nature Biotechnology, 28:1291-1294 (2010).

Methods according to this aspect of the invention may include thefollowing steps: (a) providing a mixture of nucleic acid moleculessynthesized to have the same nucleotide sequence, (b) separating nucleicacid molecules in the mixture such that amplification results in progenynucleic acid molecules being derived from a single starting nucleic acidmolecule, (c) sequencing more than one amplified nucleic acid moleculegenerated in step (b), and (d) identifying at least one individualnucleic acid with the desired sequence from the nucleic acid moleculessequenced in step (c). The nucleic acid molecule identified in step (d)may then be used as one nucleic acid molecule in an assembly process, asdescribed elsewhere herein.

According to various embodiments described herein, a computer-readablemedium may be encoded with processor-executable instructions for: (a)providing a mixture of nucleic acid molecules synthesized to have thesame nucleotide sequence, (b) separating nucleic acid molecules in themixture such that amplification results in progeny nucleic acidmolecules being derived from a single starting nucleic acid molecule,(c) sequencing more than one amplified nucleic acid molecule generatedin step (b), and (d) identifying at least one individual nucleic acidwith the desired sequence from the nucleic acid molecules sequenced instep (c). The nucleic acid molecule identified in step (d) may then beused as one nucleic acid molecule in an assembly process, as describedelsewhere herein. In various embodiments, the computer-readable mediummay be included in a system configured to reduce error from chemicallysynthesized nucleic acid molecules by selection of nucleic acidmolecules having correct nucleotide sequences.

Large nucleic acid molecules are relatively fragile and, thus, shear,readily. One method for stabilizing such molecules is by maintainingthem intracellularly. Thus, in some aspects, the invention involves theassembly and/or maintenance of large nucleic acid molecules in hostcells.

One group of organisms known to perform homologous recombination fairlyefficient is yeasts. Thus, host cells used in the practice of theinvention may be yeast cells (e.g., Saccharomyces cerevisiae,Schizosaccharomyces pombe, Pichia, pastoris, etc.).

Yeast hosts are particularly suitable for manipulation of donor genomicmaterial because of their unique set of genetic manipulation tools. Thenatural capacities of yeast cells, and decades of research have createda rich set of tools for manipulating DNA in yeast. These advantages arewell known in the art. For example, yeast, with their rich geneticsystems, can assemble and re-assemble nucleotide sequences by homologousrecombination, a capability not shared by many readily availableorganisms. Yeast cells can be used to clone larger pieces of DNA, forexample, entire cellular, organelle, and viral genomes that are not ableto be cloned in other organisms. Thus, in some embodiments, theinvention employs the enormous capacity of yeast genetics generate largenucleic acid molecules (e.g., synthetic genomics) by using yeast as hostcells for assembly and maintenance.

Exemplary of the yeast host cells are yeast strain VL6-48N, developedfor high transformation efficiency parent strain: VL6-48 (ATCC NumberMYA-3666TM)), the W303a strain, the MaV203 strain (Life TechnologiesInc., cat. no. 11281-011), and recombination-deficient yeast strains,such as the RAD54 gene-deficient strain, VL6-48-Δ54G (MATαhis3-Δ200trp1-Δ1 ura3-52 lys2 ade2-101 met14 rad54-Δ1::kanMX), which can decreasethe occurrence of a variety of recombination events in yeast artificialchromosomes (YACs).

There is a large set of selectable markers (e.g., URA3, HIS3, etc.) forselection and counter-selection of yeast mutants, making it possible tocarry out multiple rounds of seamless nucleic acid alterations withinyeast host cells. Thus, yeast can be used to introduce a number ofdifferent genetic modifications, including single nucleotide changes(e.g., insertions, deletions, mutations), modification of target nucleicacid portions and regions, and construction of entirely new chromosomes.Serial modifications to a cloned copy of an otherwise intractable genomeor other large nucleic acid can be performed in yeast in rapidsuccession. The mating capacity of yeast is favorable for modifyinggenomes and other large nucleic acids. Yeast recombination machinery,when activated during yeast mating, can be used to generate libraries,e.g., combinatorial libraries containing variants of cloned genomes ornucleic acids.

For example, Yeast Artificial Chromosome (YAC) libraries have beenconstructed for several different bacteria (Azevedo et al., PNAS USA 90,6047 (1993); Heuer et al., Electrophoresis 19, 486 (1998); Kuspa et al.,PNAS USA 86, 8917 (1989). Large prokaryotic DNA segments can be clonedin yeast using the universal genetic code. Toxic gene expressiontypically is not a barrier to cloning nucleic acids in yeast. Studieswith bacterial and archeal genomes, for example, indicate that becauseeukaryotes use different protein expression machinery than thesebacteria, there is little risk of harm to yeast hosts by proteinsexpressed from the cloned genomes. Thus, the invention further includesmethods for the generation of nucleic acid molecules (e.g., syntheticgenomes) which confer a toxic phenotype when introduced into non-yeastcell (e.g., bacterial cells).

The transcription (Kozak, Gene 234, 187 (1999)) and translation(Kornberg, Trends. Cell. Biol. 9, M46 (1999) signals in yeast aredifferent from those in bacteria. In fact, most prokaryotic genes likelyare not expressed in yeast. There is no restriction barrier in yeast(Belfort and Roberts, Nucleic Acids Res 25, 3379 (1997). If there is abarrier, it may be a replication barrier, rather than a gene expressionbarrier (Stinchcomb et al., PNAS USA 77, 4559 (1980)). Gene toxicity isminimized because regulation of gene expression in a eukaryote such asyeast is different from that in prokaryotes. Also, Mycoplasmas, forexample, use the codon UGA for tryptophan rather than as a translationstop signal. Thus, most Mycoplasma genes, if expressed, would producetruncated proteins in yeast. This largely avoids the possibility oftoxic gene products.

Nucleic acid molecules may be assembled from natural or syntheticfragments together with yeast vectors prior to transformation into yeastcells or simultaneously co-transformed into yeast cells. New organismsmay created by transferring these genomes or other nucleic acidmolecules, which have been optionally manipulated as desired, intocompatible recipient cells. Thus, one embodiment provides suitabletechniques for transferring genomes and other nucleic acid molecules toyeast host cells, modifying the genomes within host cells whilemaintaining their stability and integrity, and transplanting the clonedand manipulated genomes from yeast host cells back into recipient cellsthat more closely resemble original donors (e.g., organisms from whichthe nucleotides sequences were obtained), thus creating.

A commercially available product for the assembly of nucleic acidmolecules in yeast cells is the GENEART® High-Order Genetic AssemblySystems (Life Technology, Cat. No. A13286). This is a kit for thesimultaneous and seamless assembly of up to 10 DNA fragments, totalingup to 110 kilobases in length, into vectors. The system uses the abilityof yeast to take up and recombine DNA fragments with high efficiency.This greatly reduces the in vitro handling of DNA and eliminates theneed for enzymatic treatments, such as restriction and ligation, whileallowing for precise fusions of DNA sequences. The kit containsmaterials for the transformation and purification from yeast, includingyeast selective media, and competent E. coli for plasmid amplificationof correct clones.

Organisms other than yeast may be used for in vivo assembly. Forexample, it has been shown that exogenous DNA is integrated intohomologous sequences in the genome of Neurospora crassa at a frequencyof 100% in mutant strains deficient in non-homologous end joining.(Ninomiya et al., Highly efficient gene replacements in Neurosporastrains deficient for nonhomologous end-joining, PNAS, 100:12248-122532004.) Thus, the invention further includes methods involving organismsother than yeast (e.g., fungi such as N. crassa) and methods whichinvolve the suppression and/or elimination of non-homologous end joiningto increase the efficiency of homologous recombination. In essence, anycell which undergoes homologous recombination may be used to assemblenucleic acid molecules. However, cell most suitable for this aspect ofthe invention will be ones which naturally are efficient at performinghomologous recombination (e.g., yeasts) or can be altered (e.g., throughmutagenesis) to increase the frequency of which they homologousrecombination.

Assembly and maintenance of nucleic acid molecules in will often involveeither the generation of or the insertion into cells nucleic acidmolecule which contain elements such as one or more origin ofreplication (e.g., two origins of replication which are functional indifferent cell types) and one or selection marker (e.g., one or morepositive selection marker and/or one of more negative selection marker).

Nucleic acid molecules introduced into cells for assembly will normallyhave certain features which allow them to be assembled in a particularorder. One feature is terminal sequence homology between nucleic acidmolecules being assembled.

Assembled nucleic acid molecules may be introduced into other nucleicacid molecules located within a cell (e.g., a viral genome, a nucleargenome, an organelle genome, a bacterial chromosome, etc.). In suchinstances, functional elements such as origins of replication,centromeres, etc. will generally be present in the other nucleic acidmolecules located within the cell. Thus, the invention provides, inpart, compositions and methods relating to the assembly of nucleic acidmolecules and the insertion of the resulting assembly into other nucleicacid molecules.

In some instances, standard ligase based joining of partially and fullyassembled nucleic acid molecules may be employed. For example, fullyassembled nucleic acid molecule may be generated with restriction enzymesites near their termini. These nucleic acid molecules may then betreated with one of more suitably restrictions enzymes to generate, forexample, either one or two “sticky ends”. These sticky end molecules maythen be introduced into a vector by standard restriction enzyme-ligasemethods. In instances where the inert nucleic acid molecules have onlyone sticky end, ligases may be used for blunt end ligation of the“non-sticky” terminus.

Assembled nucleic acid molecules may also include functional elementswhich confer desirable properties (e.g., origins of replication,selectable markers, etc.). In many instances, the assembled nucleic acidmolecules will be assembled from multiple individual nucleic acidsegments with one of the segments being a vector (e.g., a linearvector).

Using the schematic of FIG. 8 for purposes of illustration, thisapproach may be carried out by co-transforming into the host cell, alongwith the host vector, a plurality (e.g., two, three, five, eight, ten,fifteen, twenty, thirty, etc.) of“overlapping” nucleic acid fragmentsfor which assembly is desired. In this instance, each of the fragmentscontains are two regions of homology to regions of other nucleic acidsegments introduced into the host cell. The nucleic acid segments aftertransformation into the host cell, for example by homologousrecombination through regions of homology. In the instance shown in FIG.8, the result is an assembled, closed circular nucleic acid molecule.

In one variation of the illustrative example shown in FIG. 8,overlapping fragments of a circular bacterial genome are co-transformedinto a yeast host cell along with a linear yeast vector. Again, theyeast vector contains regions of homology at its termini to portions ofthe bacterial genome. Upon introduction of the genome fragments andyeast host vector into the host cell, the fragments and vectorrecombine, thereby joining the genome fragments and host vector.

The process shown in FIG. 8 relies, in part, on selection for theassembly of a closed, circular, replicable nucleic acid molecule. Assimilar selection mechanisms is set out in U.S. Patent Publication No.2004/0219516 A1 (see, e.g., FIG. 20 of this application), the disclosureof which is incorporated herein by reference. Of course, nucleic acidmolecules assembled by methods of the invention need not always generatea closed circular nucleic acid molecules. Other nucleic acid moleculeswhich may be generated by methods of the invention include linearplasmids (e.g., plasmids which can replicate in linear form) andchromosomes.

In vivo assembly systems of the type shown in FIG. 8 may be composed oftwo core components: (1) Nucleic acid segments for assembly and (2) asuitable host cell. In certain embodiments where desired functionalelements (e.g., origins of replication, selectable markers, etc.) arenot represented in the nucleic acid segments for assembly, a vector maybe included as an additional nucleic acid segment.

Fragments to be assembled will generally contain sequences that areoverlapping at their termini. In one embodiment, the overlaps areapproximately 10 bp; in other embodiments, the overlaps may be 15, 25,50, 60, 70, 80 or 100 base pairs, etc. (e.g., from about 10 to about120, from about 15 to about 120, from about 20 to about 120, from about25 to about 120, from about 30 to about 120, from about 40 to about 120,from about 10 to about 40, from about 15 to about 50, from about 20 toabout 50, etc. base pairs). In order to avoid misassembly, individualoverlaps that should not be duplicated or closely match amongst thefragments. Since homologous recombination does not require 100% sequenceidentity between the participating nucleic acid molecules or regions,each terminus should be sufficiently different to prevent misassembly.Further, termini intended to undergo homologous recombination with eachother should share at least 900%, 93%, 95%, or 98% sequence identity.

In in vivo assembly methods, a mixture of all of the fragments to beassembled is used to transfect the host recombination and assembly cellusing standard transfection techniques. The ratio of the number ofmolecules of fragments in the mixture to the number of cells in theculture to be transfected should be high enough to permit at least someof the cells to take up more molecules of fragments than there aredifferent fragments in the mixture. Thus, in most instances, the higherthe efficiency of transfection, the larger number of cells will bepresent which contain all of the nucleic acid segments required to formthe final desired assembled nucleic acid molecule. Technical parametersalong these lines are set out in U.S. Patent Publication No.2009/0275086 A1, the disclosure of which is incorporated herein byreference.

One example of an assembly method which for joining double-strandednucleic acid molecules which do not share terminal sequence homology isshown in FIG. 5. In this embodiment, two double-stranded fragments areintroduced into a linear vector using singe-stranded “stitching nucleicacid molecules”. In a sense, this is an assembling of five nucleic acidsegments, wherein one of the segments is the vector, two of the segmentsare the two stitching nucleic acid molecules, and final two segments arethe segments are labeled Fragment 1 and Fragment 2. In addition tofacilitating the joining of other nucleic acid molecules, the stitchingnucleic acid molecules introduced short insertion (e.g., nine basepairs) into the assembled nucleic acid molecule. A commerciallyavailable product which contains these features is the GENEART®High-Order Genetic Assembly Systems (Life Technology, Cat. No. A13286).

Nucleic acid molecules may also be assembled or otherwise designed withsite specific recombination sites (e.g., GATEWAY® sites) and/ortopoisomerase sites. Site specific recombinases are recombinase whichtypically have at least the following four activities (or combinationsthereof): (1) recognition of one or two specific nucleic acid sequences;(2) cleavage of said sequence or sequences; (3) topoisomerase activityinvolved in strand exchange; and (4) ligase activity to reseal thecleaved strands of nucleic acid. (See Sauer, B., Current Opinions inBiotechnology 5:521-527 (1994)). Conservative site-specificrecombination is distinguished from homologous recombination andtransposition by a high degree of specificity for both partners. Thestrand exchange mechanism involves the cleavage and rejoining ofspecific nucleic acid sequences in the absence of DNA synthesis (Landy,A., Ann. Rev. Biochem. 58:913-949 (1989)).

One means by which nucleic acid molecules may be assembled is by the useof recombinational cloning. Thus, the invention includes compositionsand methods related to recombination cloning and recombination sites, aswell as recombination cloning components.

A number of recombinational cloning systems are known. Examples ofrecombination sites which may be sued in such systems include, but arenot limited to, loxP sites; loxP site mutants, variants or derivativessuch as loxP511 (see U.S. Pat. No. 5,851,808); frt sites; frt sitemutants, variants or derivatives; dif sites; dif site mutants, variantsor derivatives; psi sites; psi site mutants, variants or derivatives;cer sites; and cer site mutants, variants or derivatives.

These cloning systems are typically based upon the principle thatparticular recombination sites will recombine with their cognatecounterparts. Nucleic acid molecules of the invention may be designed soas they contain recombination sites of different recombinational cloningsystems (e.g., lox sites and att sites). As an example, a nucleic acidmolecule of the invention may contain a single lox site and two attsites, wherein the att sites do not recombine with each other.

Recombination sites for use in the invention may be any nucleic acidthat can serve as a substrate in a recombination reaction. Suchrecombination sites may be wild type or naturally occurringrecombination sites, or modified, variant, derivative, or mutantrecombination sites. Examples of recombination sites for use in theinvention include, but are not limited to, phage lambda recombinationsites (such as attP, attB, attL, and attR and mutants or derivativesthereof) and recombination sites from other bacteriophage such as phi80,P22, P2, 186, P4 and P1 (including lox sites such as loxP and loxP511).Mutated att sites (e.g., attB 1-10, attP 1-10, attR 1-10 and attL-1 10)are described in U.S. Appl. No. 60/136,744, filed May 28, 1999, and U.S.application Ser. No. 09/517,466, filed Mar. 2, 2000, which arespecifically incorporated herein by reference. Other recombination siteshaving unique specificity (i.e., a first site will recombine with itscorresponding site and will not recombine with a second site having adifferent specificity) are known to those skilled in the art and may beused to practice the present invention. Corresponding recombinationproteins for these systems may be used in accordance with the inventionwith the indicated recombination sites. Other systems providingrecombination sites and recombination proteins for use in the inventioninclude the FLP/FRT system from Saccharomyces cerevisiae, the resolvasefamily (e.g., TndX, TnpX, Tn3 resolvase, Hin, Hjc, Gin, SpCCE1, ParA,and Cin), and IS231 and other Bacillus thuringiensis transposableelements. Other suitable recombination systems for use in the presentinvention include the XerC and XerD recombinases and the psi, dif andcer recombination sites in Escherichia coli. Other suitablerecombination sites may be found in U.S. Pat. No. 5,851,808 issued toElledge and Liu which is specifically incorporated herein by reference.Recombination proteins and mutant, modified, variant, or derivativerecombination sites which may be used in the practice of the inventioninclude those described in U.S. Pat. Nos. 5,888,732 and 6,143,557, andin U.S. application Ser. No. 09/438,358 (filed Nov. 12, 1999), basedupon U.S. provisional application No. 60/108,324 (filed Nov. 13, 1998),and U.S. application Ser. No. 09/517,466 (filed Mar. 2, 2000), basedupon U.S. provisional application No. 60/136,744 (filed May 28, 1999),as well as those associated with the GATEWAY® Cloning Technologyavailable from Life Technologies Corp., (Carlsbad, Calif.), the entiredisclosures of all of which are specifically incorporated herein byreference in their entireties.

Representative examples of recombination sites which can be used in thepractice of the invention include att sites referred to above. Att siteswhich specifically recombine with other att sites can be constructed byaltering nucleotides in and near the 7 base pair overlap region. Thus,recombination sites suitable for use in the methods, compositions, andvectors of the invention include, but are not limited to, those withinsertions, deletions or substitutions of one, two, three, four, or morenucleotide bases within the 15 base pair core region (GCTTTTTTATACTAA(SEQ ID NO: 2)), which is identical in all four wild-type lambda artsites, attB, attP, attL and attR (see U.S. application Ser. No.08/663,002, filed Jun. 7, 1996 (now U.S. Pat. No. 5,888,732) and Ser.No. 09/177,387, filed Oct. 23, 1998, which describes the core region infurther detail, and the disclosures of which are incorporated herein byreference in their entireties). Recombination sites suitable for use inthe methods, compositions, and vectors of the invention also includethose with insertions, deletions or substitutions of one, two, three,four, or more nucleotide bases within the 15 base pair core region(GCTTTTTTATACTAA (SEQ ID NO: 2)) which are at least 50% identical, atleast 55% identical, at least 60% identical, at least 65% identical, atleast 700/% identical, at least 75% identical, at least 80% identical,at least 85% identical, at least 90% identical, or at least 95%identical to this 15 base pair core region.

Analogously, the core regions in attB1, attP1, attL1 and attR1 areidentical to one another, as are the core regions in attB2, attP2, attL2and attR2. Nucleic acid molecules suitable for use with the inventionalso include those which comprising insertions, deletions orsubstitutions of one, two, three, four, or more nucleotides within theseven base pair overlap region (TTTATAC, which is defined by the cutsites for the integrase protein and is the region where strand exchangetakes place) that occurs within this 15 base pair core region(GCTTTTTTATACTAA (SEQ ID NO: 2)).

Multi-Site GATEWAY® technology is described in U.S. Patent PublicationNo. 2004/0229229 A1, the entire disclosure of which is incorporatedherein by reference, and is effective for cloning multiple DNA fragmentsinto one vector without using restriction enzymes. This system can beused to link 1, 2, 3, 4, 5 or more nucleic acid segments, as well as tointroduce such segments into vectors (e.g., a single vector). TheGATEWAY® (e.g., Multi-Site GATEWAY®) system allows for combinations ofdifferent promoters, DNA elements, and genes to be studied in the samevector or plasmid, for efficient gene delivery and expression. Insteadof transfecting multiple plasmids for each gene of interest, a singleplasmid carrying different DNA elements, referred to as “an expressioncassette” can be studied in the same genomic background.

The present invention also relates to methods of using one or moretopoisomerases to generate assembled nucleic acid molecules.Topoisomerases may be used in combination with recombinational cloningtechniques described herein. For example, a topoisomerase-mediatedreaction may be used to attach one or more recombination sites to one ormore nucleic acid segments. The segments may then be further manipulatedand combined using, for example, recombinational cloning techniques.

In one aspect, the present invention provides methods for linking afirst and at least a second nucleic acid segment topoisomerase (e.g., atype IA; type IB, such as Vaccinnia virus topoisomerase; and/or type IItopoisomerase) such that either one or both strands of the linkedsegments are covalently joined at the site where the segments arelinked.

A method for generating a double stranded recombinant nucleic acidmolecule covalently linked in one strand can be performed by contactinga first nucleic acid molecule which has a site-specific topoisomeraserecognition site (e.g., a type IA or a type II topoisomerase recognitionsite), or a cleavage product thereof, at a 5′ or 3′ terminus, with asecond (or other) nucleic acid molecule, and optionally, a topoisomerase(e.g., a type IA, type IB, and/or type II topoisomerase), such that thesecond nucleotide sequence can be covalently attached to the firstnucleotide sequence. As disclosed herein, methods of the invention canbe performed using any number of nucleotide sequences, typically nucleicacid molecules wherein at least one of the nucleotide sequences has asite-specific topoisomerase recognition site (e.g., a type IA, type IBor type II topoisomerase), or cleavage product thereof, at one or both5′ and/or 3′ termini.

Topoisomerase mediated nucleic acid ligation methods are described indetail in U.S. Patent Publ. No. 2004/0265863 A1, the entire disclosureof which is incorporated herein by reference.

Assembled nucleic acid molecules may be cloned may contain a blunt endto be linked, and the second nucleic acid molecule involved in thecloning method may contain an overhang at the end which is to be linkedby a site-specific topoisomerase (e.g., a type IA or a type IBtopoisomerase), wherein the overhang includes a sequence complementaryto that comprising the blunt end, thereby facilitating strand invasionas a means to properly position the ends for the linking reaction.

Any number of vectors may be used in the practice of the invention.Further, the selection of vectors for particular applications will varywith the specifics of those applications (e.g., the host cell). In manyinstances, vectors will be introduced into host cells in linear form.

Suitable vectors for use in the present invention also includeprokaryotic vectors such as pcDNAII, pSL301, pSE280, pSE380, pSE420,pTrcHisA, B, and C, pRSET A, B, and C (Life Technologies Corp.),pGEMEX-1, and pGEMEX-2 (Promega, Inc.), the pET vectors (Novagen, Inc.),pTrc99A, pKK223-3, the pGEX vectors, pEZZ18, pRIT2T, and pMC1871(Pharmacia, Inc.), pKK233-2 and pKK388-1 (Clontech, Inc.), and pProEx-HT(Life Technologies Corp.) and variants and derivatives thereof. Othervectors of interest include eukaryotic expression vectors such aspFastBac, pFastBacHT, pFastBacDUAL, pSFV, and pTet-Splice (LifeTechnologies Corp.), pEUK-CI, pPUR, pMAM, pMAMneo, pBI101, pBI121, pDR2,pCMVEBNA, and pYACneo (Clontech), pSVK3, pSVL, pMSG, pCH110, andpKK232-8 (Pharmacia, Inc.), p3′SS, pXT1, pSG5, pPbac, pMbac, pMC1neo,and pOG44 (Stratagene, Inc.), and pYES2, pAC360, pBlueBacHis A, B, andC, pVL1392, pBlueBacIII, pCDM8, pcDNA1, pZeoSV, pcDNA3 pREP4, pCEP4, andpEBVHis (Life Technologies Corp.) and variants or derivatives thereof.

Other vectors suitable for use in the invention include pUC18, pUC19,pBlueScript, pSPORT, cosmids, phagemids, YAC's (yeast artificialchromosomes), BAC's (bacterial artificial chromosomes), P1 (Escherichiacoli phage), pQE70, pQE60, pQE9 (Qiagen), pBS vectors, PhageScriptvectors, BlueScript vectors, pNH8A, pNH16A, pNH18A, pNH46A (Stratagene),pcDNA3 (Life Technologies Corp.), pGEX, pTrsfus, pTrc99A, pET-5, pET-9,pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia), pSPORT1, pSPORT2,pCMVSPORT2.0 and pSV-SPORT1 (Life Technologies Corp.) and variants orderivatives thereof.

Assembly methods, in addition to other methods described herein, arecapable of being miniaturized and/or automated. In fact, in manyinstances, miniaturization will be desirable when the nucleic acidmolecules being assembled and/or introduced into vectors are present inlower total numbers. One means by which micro-mixing can be accomplishedfor assembly and processes such as insertion of nucleic acid moleculesinto vectors is by electrowetting, for example, as described elsewhereherein.

FIG. 16 is a block diagram of one embodiment of an instrument forprocessing nucleic acid molecules of the invention. On the upper left ofthis figure is a carrier oil reservoir 1600 and a tube 1601 fortransporting oil from this reservoir. Carrier oil is transported past aseries of additional reservoirs 1602 that contain reagents. The circularstructures represent individual reagents reservoirs. Exemplary reagentsare nucleic acid molecules, PCR enzymes, primers, and vectors, as wellas, for example, other Module 3 related components. The reagents willtypically be in the form of aqueous vesicles transported between oilbarriers.

The reagents transported by the carrier oil are then transported to amixing chamber 1603 where mixing occurs. The reagents then move on to adigital PCR station 1604. The tube 1601 travels between a heating block1605 where denaturation occurs followed by a cooling block 1606 whereannealing and PCR occurs. Each time a vesicle travels to the coolingblock 1606 after the first time, a PCR amplification occurs.

After exiting the digital PCR station 1604, the vesicles move pastadditional reagent reservoirs 1607 for the optional addition of morereagents, (e.g., buffers, error correction components, etc.), then on toanother optional mixing chamber 1608. The vesicle then move on to anoptional storage location 1609. In instances where more than one nucleicacid molecule is to be assembled into a larger molecule, the individualnucleic acid molecules for assembly will often arrive at the storagelocation 1609 at different times and will need to be sequestered for aperiod of time until other components arrive.

The nucleic acid molecules then move on to another digital PCR station1610 and again cycle between cooling and heating blocks. Errorcorrection reaction may occur in digital PCR station 1610. Finally,assembled nucleic acid molecules are transported to interface outlets1611 for collection and waste materials (e.g., carrier oil) iscollection in a waste reservoir 1612.

Systems of the type represented in FIG. 16 can process multiple samplesat the same time. These samples can be sequestered between carrier oiland sent through the system in series. The FIG. 16 block diagram doesnot show a computer system, optical components, valves and othercomponents related to system automation. Optical elements, as well asother elements (e.g., electrical elements) can be used to keep track ofthe location and identification of reagent vesicles and various pointsin the flow system. These reagent vesicles will generally containnucleic acid molecules of a predetermined sequence. Thus, the inventioninclude methods for the simultaneous processing (e.g., sequentialprocessing) of multiple samples

Module 4

Following isolation and treatment, the assembled nucleic acid moleculescan be further transplanted into recipient cells using methods describedherein or known in the art. Methods which may be used include protoplastand spheroplast fusion, conjugal transfer (e.g., bacterial conjugation),viral infection, electroporation and Sendai virus mediated cell fusion.Thus, the invention includes methods for transferring synthesized and/orassembled nucleic acid molecules to cells.

One method for generating yeast protoplast fusions in set out inNakazawa and Iwano, Efficient selection of hybrids by protoplast fusionusing drug resistance markers and reporter genes in Saccharomycescerevisiae, J. Biosci. Bioeng. 98:353-358 (2004). Further, methods havebeen developed for the fusion or prokaryotic and eukaryotic cells. (See,e.g., Gyuris and Duda, High-efficiency transformation of Saccharomycescerevisiae cells by bacterial minicell protoplast fusion, Mol. Cell.Biol. 6:3295-3297 (1986). Methods such as these may be used in thepractice of the invention to transfer nucleic acid molecules betweencells without exposing the nucleic acid molecules to an extracellularenvironment. Other methods which may be used include natural competence,biolistic gun, electroporation, Baculovirus mediated transduction, andType III secretion systems.

An exemplary transplantation protocol is described in PCT Publication WO2011/109031. One method used to transplant Mycoplasma genomes fromdonors to Mycoplasma recipients is described by Lartigue et al., Genometransplantation in bacteria: changing one species to another, Science317:632 (2007). This work related to the complete replacement of thegenome of a bacterial cell with a genome from another species by genometransplantation as naked DNA using polyethylene glycol-mediatedtransformation. The resulting recipient cells were phenotypicallyidentical to the donor strain. Such methods can be used to transferassembled nucleic acid molecules constructed by methods of the inventionto recipient cells.

Recipient cells typically will be chosen based on their ability tosupport gene expression from the assembled nucleic acid molecules. Forexample, after a bacterial genome has been assembled in a eukaryotichost cell having a suitable genetic manipulation system (e.g., yeast),then it may be necessary or desirable to transplant the genome back intoa bacterial recipient cell. Differences in translation and transcriptionand different codon usage, among other factors, can prevent expressionof the donor gene products within the host cell. The recipient cell,therefore, may be of the same species or a similar species as a donorcell or organism. In many cases, the recipient cells will be of the sameorder or kingdom as the donor. However, in cases where expression inunrelated cell types is required, the initial gene design may includecodon and sequence optimization strategies to allow for expression indifferent recipient cells.

Following isolation of donor nucleic acids in agarose plugs, host DNAcan optionally be removed (e.g., by digest and/or electrophoresis), andoptionally treated with methyltransferases and/or proteinase.

Agarose plugs can be melted, for example, by incubation with β-Agarase I(New England Biolabs) as described in Example 3A(ii)(b) of PCTPublication WO 2011/109031.

Transplantation can be performed in the presence of polyethylene glycol(PEG), such as PEG-6000 or PEG-8000 or other PEG to facilitatetransformation. The source, amount, and size of the PEG can be varied todetermine the optimal PEG. In one example, the PEG is PEG-2000,PEG-40000 PEG-6000, PEG-8000, PEG-10000, PEG-20000, or other. Theconcentration of PEG can be varied depending upon the conditions of thetransplantation; concentrations include those, for example, at or about5% or at or about 10%. An example is described in Example 3A(ii)(c) ofPCT Publication WO 2011/109031. Melted plugs can be added to therecipient cells in the presence of PEG with gentle rocking to mix. Cellsare allowed to recover, centrifuged, and grown in medium containingappropriate selection medium to select for recipient cells containingthe transplanted donor nucleic acid. In one aspect, cells are plated onthe medium and grown under appropriate conditions for the recipient celltype until colonies appear. Colonies can be picked and further grown inselection medium to produce a desired quantity of recipient cellscontaining the transplanted genome or other donor nucleic acid.

A particular ratio of recipient cells to donor nucleic acid can bemaintained as needed. In one example, a ratio of between at or about 10⁷and at or about 10⁸ recipient cells per 2 μg genomic DNA can bemaintained. The provided transplantation methods can be used to achieveapproximately 30 transformants for 200 ng of endogenous genomic DNA, orbetween 500 and 1500 transplants per reaction, or other appropriateamount that is obtained from the host or donor cell. In one non-limitingexample, transplantation is carried out with 10⁷ recipient cells, 20picoliters of melted of agarose plug containing donor genome at 100ng/μl. One would understand that the ratio of recipient cells to donornucleic acid may vary depending upon the cell types and that empiricalassessment can be used to optimize the ratio.

Selection of recipient cells which contain a transplanted donor nucleicacid can be performed by any number of means. For example, transplanteddonor nucleic acid may contain a positive selection marker which willallows it to be maintained in recipient cells. Also, acounter-selectable marker may be introduced in the recipient cell genometo allow for selection against cells which retain these nucleic acidmolecules. A combination of positive and counter selection can beemployed if one desires an engineered recipient cell which contains atransplanted donor nucleic acid but not the original recipient cellgenome.

Further, in some embodiment, a plurality (e.g., two, three, four, five,etc.) of donor nucleic acid molecules (e.g., genomes from differentorganisms), may be introduced into a single host cell. For example, adiploid yeast strain containing genomes of two different organisms, suchas two Mycoplasma genomes from different species, can be generated bycrossing two different haploid strains, each carrying one of thegenomes. Crossing haploid yeast strains can be carried out usingwell-known methods.

Multiple distinct selection markers can be used in the respectivehaploid strains, to allow for selection of cells containing both genomesafter the cross. For example, a HIS3 and TRP marker can be introducedinto two different haploid cells, respectively, carrying differentgenomes, followed by selection of diploid cells on medium lackinghistidine and tryptophan, as described in the Examples of PCTPublication WO 2011/109031.

Assembled nucleic acid molecules may be used for any number of purposes.For example, in many instances, it will be desirable to introduce suchmolecules into cells for particular applications. The components of theassembled nucleic acid molecules and the cells that they are introducedinto will vary widely with the particular application.

One illustration of an application is a prokaryotic production cell linefor which an assembled nucleic acid molecule represents the entiregenome. This genome may be designed for minimal functionality with thefollowing features represent/absent:

1. Lack of ability to undergo conjugation or mating (safety feature).

2. Lack of ability to synthesize a critical nutrient (safety feature).

3. Maintain a high energy charge (production efficiency feature).

4. A pathway for generation of a desired end product (productionfeature).

While features included or excluded from cells generated by methods ofthe invention can vary greatly, in many instances, safety features willbe included to prevent “escape” of the organism and limit the ability ofthe organisms to transfer traits to other organisms. Production featuresmay be included to tailor the organisms for a specific application. Thistailoring may fine tuned in a manner not currently possible with a“chasis” organisms. A chasis organism is an organism which has many ofthe features the desired application but requires modification to makeit fully suitable. Typically this modification results from (1) theinactivation of one or more gene and/or pathway and/or (2) theintroduction of one or more gene. In some instances, assembled nucleicacid molecules may be introduced into a chasis organism with or withoutthe ultimate elimination of the chasis organism genome.

A recipient cell can be, for example, a bacterial cell, a yeast cell, afungal cell, an insect cell, a mammalian cell, a plant cell or an algalcell.

The invention includes methods for producing nucleic acid molecules(e.g., individual coding elements, genomes, etc.) designed to yield highlevel production of desired end products, as well as the nucleic acidmolecules themselves and organisms into which these nucleic acidmolecules are introduced. Using amino acid biosynthesis for purposes ofillustration, many organisms can produce lysine on their own but do soin limited quantities. In many instances, L-aspartate is a startingcompound for L-lysine production. Further, amino acids which may beproduced as part of the conversion of aspartate to lysine includeL-threonine, L-methionine and L-isoleucine. Further, a number of enzymesare involved in the conversion of aspartate to lysine, often startingwith aspartate kinase. As would be apparent to one skilled in the art,pathways associated with the synthesis of L-lysine may also be alteredfor high level production of L-threonine, L-methionine and/orL-isoleucine. Enzymes involved in the production of L-lysine,L-threonine, L-methionine and L-isoleucine are set out in U.S. Pat. No.7,323,321, the entire disclosure of which is incorporated herein byreference.

Pathway engineering can be employed to introduce constitutive, inducibleand repressible promoters at specific points in the metabolic pathway todrive production towards a designed end product (e.g., L-lysine).Pathway engineering will often be employed in a manner that allows forthe direction of cellular resources (e.g., energy charge, nutrients) ofa cell to be directed to two functions: (1) Cell growth/division and (2)end product production. Thus, in some embodiments, the inventionincludes methods for designing and constructing cells, as well as thecells themselves, that channel cellular resources into two functions:(1) Cell growth/division and (2) end product production.

Cells of the invention may be designed to not engage in activitiesnormally associated with wild-type cells. One of these activities ismating. Mating consumes cellular resources and facilitates genetransmission. In many instances, neither of these effects of mating willbe desirable. Further, in some instances, mating leads to sporulation.Spore formatting may be desirable for storage of organisms but, in manyinstances, if sporulation is desired, then mating genes may be placedunder tight regulatory control or instructed on vectors.

In some embodiments, the invention includes cells designed andconstructed to have a minimal genome for the desired purpose. DNAreplication, transcription, and translation, as examples, consumecellular resources. Thus, one method for providing for efficientcellular resource channeling is to design and/or use a cell with aminimal genome.

With respect to cell division, if basic molecules required for cellularfunction are decreased below certain levels, then cell growth anddivision will generally be impacted. Again using amino acid productionfor purposes of illustration, when lysine is the desired end productthere are at least three choices for providing suitable concentrationsof threonine, methionine and isoleucine for cellular metabolism: (1)Allowing for production of these amino acids by alternative pathways,(2) using promoters which allow for some production of these amino acidsas side products of lysine production, and (3) supplying these aminoacid exogenously.

The invention thus include methods for pathway engineering of cell, suchmethods comprising:

(a) synthesizing a plurality of nucleic acid molecules, wherein eachnucleic acid molecule is prepared in a microquantity;

(b) joining some or all of the nucleic acid molecules present in thepool formed in (a) to form a plurality of larger nucleic acid molecules;and

(c) assembling the plurality of larger nucleic acid molecules to formthe nucleic acid molecule which encodes at least two expressionproducts,

wherein at least two of the at least two expression products are in thesame biological pathway that converts a starting compound (e.g.,L-aspartate) to a desired end product (e.g., L-lysine, L-isoleucine,etc.).

Various embodiments of the invention include computer-implementedmethods for pathway engineering of cell. These methods may beimplemented by a processor by executing instructions encoded on acomputer-readable medium. According to various embodiments, theinstructions may be for:

(a) synthesizing a plurality of nucleic acid molecules, wherein eachnucleic acid molecule is prepared in a microquantity;

(b) joining some or all of the nucleic acid molecules present in thepool formed in (a) to form a plurality of larger nucleic acid molecules;and

(c) assembling the plurality of larger nucleic acid molecules to formthe nucleic acid molecule which encodes at least two expressionproducts,

wherein at least two of the at least two expression products are in thesame biological pathway that converts a starting compound (e.g.,L-aspartate) to a desired end product (e.g., L-lysine, L-isoleucine,etc.).

One application of technology of the invention is in biofuel production.In many instances, this involves the conversion of a carbon source to abiofuel or a biofuel precursor. Biofuel or biofuel precursors varywidely, as do cell suitable for their production. In many instances,cells used for the production of biofuel or biofuel precursors will bealgal or plant cells. Exemplary algae which may be used in this andother aspects of the invention include Anabaena sp., Chlamydomonasreinhardtii, Chlorella sp., Cyclotella sp., Gloeobacter violaceus,Nannochloropsis sp., Nodularia sp., Nostoc sp., Prochlorococcus sp.,Synechococcus sp., Oscillatoria sp., Arthrospira sp., Lyngbya sp.,Dunaliella sp., and Synechocystis sp.

Many species of plants may be cultivated from a single or small numberof plant cells. Thus, plants which contain assembled nucleic acidmolecules in most, if not all of their cells, may be generated.Exemplary algae which may be used in this and other aspects of theinvention include corn, soybeans, rapeseed, sugar cane, mustard,switchgrass, and jatropha.

Biofuels, biofuel precursors and related compounds produced applicationsmay be useful for applications which include the following: spaceheating, lighting, cooking, and running of automobile engines andgenerators.

Exemplary biofuels and biofuel precursors include normal-chain alcohols(the alcohol group —OH attached to the terminal carbon) having greaterthan 3 carbon atoms up to 21 carbon on. Normal chain alcohols, which maybe produced by methods of the invention, include n-butanol, n-pentanol,n-hexanol, n-heptanol, n-octanol (capryl alcohol), n-nonanol (pelargonicalcohol), n-decanol (capric alcohol), n-dodecanol (lauryl alcohol),n-pentadecanol, n-hexadecanol (cetyl alcohol), n-tetradecanol (myristylalcohol), cis-9-hexadecen-1-ol (palmitoleyl alcohol), n-octadecanol(stearyl alcohol), 9E-octadecen-1-ol (elaidyl alcohol),cis-9-octadecen-1-ol (oleyl alcohol), 9Z, 12Z-octadecadien-1-ol(linoleyl alcohol), 9E, 12E-octadecadien-1-ol (elaidolinoleyl alcohol),9Z, 12Z, 15Z-octadecatrien-1-ol (linolenyl alcohol), 9E, 12E,15-E-octadecatrien-1-ol (elaidolinolenyl alcohol),12-hydroxy-9-octadecen-1-ol (ricinoleyl alcohol) and 1-eicosanol(arachidyl alcohol or combinations thereof. Normal chain alcohols may besaturated or unsaturated.

n-butanol can be produced by microbial fermentation, chemicallysynthesized or obtained from a plant source by bacterial action (e.g.,engineered bacterial generated by methods of the invention). Thisincludes obtaining butanol from cellulose containing plants,lignin-containing plants, from sewage and animal waste, from sugarsobtained from plant source and then by fermentation involving algae(e.g., engineered algae generated by methods of the invention). Higheralcohols can also be obtained in similar manner.

The invention may also be used to produce chemical intermediates.Example of such intermediates are 1,4-butanediol and 1,3-propanediol.1,4-butanediol is a bifunctional alcohol with a broad array of uses inthe chemical industry. As examples, butanediol and its derivatives areused in the production of plastics, polyurethanes, solvents, electronicchemicals and elastic fibers. A 1,4-butanediol synthesis pathway is setout in Burk, International Sugar Journal 112:30-35 (2010). Thus, theinvention includes cells engineered to produce or for increasedproduction of chemical intermediates (e.g., 1,4-butanediol,1,3-propanediol, etc.), as well as methods for designing and producingsuch cells.

Additional Applications

As one skilled in the art would understand, nucleic acid moleculesproduced in microscale quantities (e.g., femtomoles to nanomolesquantities, such as from about 0.001 femptomole to about 1.0 nanomole,from about 0.01 femptomole to about 1.0 nanomole, from about 0.1femptomole to about 1.0 nanomole, from about 0.001 femptomole to about0.1 nanomole, from about 0.001 femptomole to about 0.01 nanomole, fromabout 0.001 femptomole to about 0.001 nanomole, from about 1.0femptomole to about 1.0 nanomole, from about 1.0 femptomole to about 0.1nanomole, from about 1.0 femptomole to about 0.01 nanomole, from about1.0 femptomole to about 0.001 nanomole, from about 10 femtomoles toabout 1.0 nanomole, from about 10 femtomoles to about 0.001 nanomole,from about 20 femtomoles to about 1.0 nanomole, from about 100femtomoles to about 1.0 nanomole, from about 500 femtomoles to about 1.0nanomole, from about 1 nanomole to about 800 nanomoles, from about 40nanomoles to about 800 nanomoles, from about 100 nanomoles to about 800nanomoles, from about 200 nanomoles to about 800 nanomoles, from about500 nanomoles to about 800 nanomoles, from about 100 nanomoles to about1,000 nanomoles, etc.).

The invention may be used to prepare microarrays. Such microarrays maybe generated in multiple ways including by the depositing of nucleicacid molecules on a support (e.g., a solid support such as a planar soldsupport) or by synthesis of nucleic acid directly on the support. In oneembodiment, the plate shown in FIGS. 2A-2B can be modified so that thebase/bottom is designed for the synthesis of nucleic acid on itssurface. Optionally, the base could be structured to be removable toyield, for example, a planar microarray. In most such instances, thebead shown in FIGS. 2A-2B would be omitted during nucleic acidsynthesis. Thus, the invention includes methods for the generation ofmicroarrays.

Methods for printing microarrays are set out in U.S. Pat. Nos. 5,807,522and 7,211,148, the disclosure of which is incorporated herein byreference. Such methods may be used in the practice of the invention toproduce, for example, microarrays by the deposition of nucleic acidmolecules produced as described herein.

One advantage of methods described herein is their modularity. As anexample, nucleic acid molecules which form sub-portions of differentlarger nucleic acid molecules may be produced on the same plate toarray. Thus, methods of the invention allow for the simultaneousproduction of nucleic acid molecules, followed by selection ofindividual synthesized nucleic acid molecules for later processes (e.g.,pooling, cleavage deprotection, and assembly). Thus, methods of theinvention include those where nucleic acid molecules are simultaneouslyproduced (e.g., chemically synthesized), followed by assembly into twoor more (e.g., two to ten, three to ten, four to ten, five to ten, twoto thirty, five to thirty, ten to thirty, five to fifty, etc.) largernucleic acid molecules.

In certain embodiments, nucleic acid molecules or plurality of nucleicacid molecules synthesized by the methods of the present invention maybe primers and/or probes. Primers and/or probes can be generated inmicroquantity using, for example, a solid support as described herein.Primers prime nucleic acid extension reactions that can be part of anamplification reaction. Probes are used to detect a target nucleic acidsequence. Accordingly, probes are used in detection methods to directlyor indirectly detect a target nucleic acid sequence. Primers and probestypically have a predetermined nucleotide sequence that hybridize withor otherwise bind to a target nucleic acid sequence. Probes inillustrative embodiments include a label, such as a fluorescent label.For example, a control mechanism may be connected to a solid support oran array of solid supports used in the methods of the present invention,wherein a target nucleotide sequence is input into the controlmechanism. The control mechanism may be used to direct the sequence ofaddition of reactants for nucleic acid synthesis, such that a nucleicacid molecule having the target nucleotide sequence is synthesized.

Probes and primers hybridize with or otherwise bind to a target nucleicacid sequence because of sequence identity they share with the targetnucleic acid sequence. For example, a primer or probe can share 80, 85,90, 95, 96, 97, 98, 99, 99.5, or 100%/o contiguous sequence identitywith a target nucleic acid sequence. Primers and probes hybridize withtheir target nucleic acid sequence under stringent and typically highlystringent conditions, as are known in the art.

A label can be attached to the 5′ terminal nucleotide, the 3′ terminalnucleotide, or any internal nucleotide of the primers and/or probes ofthe present invention. The label in certain illustrative embodiments, isa fluorophore. A vast array of fluorophores are known to those of skillin the art and can be included in the methods and compositions of thepresent invention. See, for example, Cardullo et al, Proc. Natl. Acad.Sci. USA 85:8790-8794 (1988); Dexter, D. L, J. of Chemical Physics21:836-850 (1953); Hochstrasser et al., Biophysical Chemistry 45:133-141(1992); Selvin, R, Methods in Enzymology 246:300-334 (1995); Steinberg,I., Ann. Rev. Biochem, 40:83-114 (1971); Stryer, L., Ann. Rev. Biochem,47:819-846 (1978); Wang et al., Tetrahedron Letters 31:6493-6496 (1990);Wang et al., Anal. Chem. 67:1197-1203 (1995). For example, thefluorophore can be Biosearch Blue, FAM, TET, a CAL Fluor dye, JOE, VIC,HEX, a Quasar dye, a Cy dye, NED, TAMRA, ROX, Texas Red, or a Pulsardye. These dyes and nucleic acid synthesis reactants that include thesedyes are commercially available, for example, from BiosearchTechnologies, Inc., Glen Research, or Life Technologies.

In illustrative embodiments, primers synthesized by methods providedherein, are PCR primers. In certain embodiments, primers are labeledwith a label on their 5′ end or 3′ end. For example, primers can be LUXprimers, Scorpion primers, Amplifluor primers, and/or Plexor primers.

In certain embodiments, the present invention provides a method forsynthesizing a plurality of primer and probe sets (e.g., pairs). Theprimer and probe sets (e.g., pairs) can be generated in microquantityusing a plate described herein (e.g., a plate of the general formatshown in FIGS. 2A-2B). A primer and probe set (e.g., pair) includes oneor more primers that prime an extension reaction that generates anucleic acid extension product that is a target nucleic acid sequencefor one or more probes of the primer and probe set (e.g., pair). Inother words, in a primer and probe set (e.g., pair), the probe typicallybinds to the amplification product generated by the primer(s). Inillustrative embodiments, the primer and probe set (e.g., pair) includea pair of PCR primers and a probe that binds to an amplification productgenerated by an amplification reaction that uses the pair of primers.For example, the primer and probe set (e.g., pair) can include two PCRprimers and one 5′ nuclease probe or one Molecular Beacons probe thatbinds to the amplification product generated when the PCR primers areused in a PCR reaction.

As noted above, methods of the present invention can generate an arrayof nucleic acid molecules, such as primers, probes, and/or primer andprobe sets (e.g., pairs). For example, nucleic acid molecules can besynthesized in an array of positions such that each position includesone or a plurality of nucleic acid molecules such as primers, probes,and/or primer and probe sets (e.g., pairs). Array can include primers,probes, and primer and probe sets (e.g., pairs) at a density of 100,200, 250, 500, 1000, 10,000, 100,000, 1,000,000, or 10,000,000 per cm².The total number of nucleic acid molecules in an array of nucleic acidmolecules generated using methods of the present invention can include,for example, 100, 200, 250, 500, 1000, 10,000, 100,000, 1,000,000,10,000,000, 100,000,000, 1,000,000,000, or 10,000,000,000 primer,probes, and/or primer and probe sets (e.g., pairs). More than one primerand probe set (e.g., pair) can be included in an array position suchthat the primer and probe set (e.g., pair) are designed to perform amultiplex reaction, such as a multiplex PCR reaction.

Probes of the invention can be labeled with a single dye, such as asingle fluorophore. Probes of the invention can be FISH probes.

Probes of the invention can be probes used in amplification reactions.For example, these probes can be dual-labeled probes. Dual-labeledprobes in certain illustrative embodiments include labels that aredonor-acceptor energy transfer pairs, such as FRET pairs. When the donor(fluorophore) is a component of a probe that utilizes donor-acceptorenergy transfer, the donor fluorescent moiety and the quencher(acceptor) of the invention are preferably selected so that the donorand acceptor moieties exhibit donor-acceptor energy transfer when thedonor moiety is excited. One factor to be considered in choosing thefluorophore-quencher pair is the efficiency of donor-acceptor energytransfer between them. In many instances, the efficiency of FRET betweenthe donor and acceptor moieties is at least 10%, at least 50%, or atleast 80%. The efficiency of FRET can easily be empirically tested usingthe methods both described herein and known in the art.

In some instances, the donor-acceptor pair may include a fluorophore anda quencher. The quencher can be a dark quencher. As such, probes of thepresent invention can include a BHQ dye or a DQ dye (Epoch) as thequencher. The quencher in other embodiments may be DABCYL or TAMRA.

Primers and probes synthesized using methods and systems of the presentinvention can include can include moieties that stabilize hybridizationof nucleic acids (e.g., intercalators, minor groove binding moieties,bases modified with a stabilizing moiety (e.g., alkynyl moieties, andfluoroalkyl moieties)), and conformational stabilizing moieties, such asthose disclosed in U.S. Patent Application Publication No. 2007/0059752,the disclosure of which is incorporated herein by reference. The primersand probes can include intercalating agents such as acridine. In otherembodiment, primers and probes synthesized using methods and systems ofthe present invention can be locked nucleic acid (LNA) probes, orpeptide nucleic acid (PNA) probes.

Dual-labeled probes synthesized using methods and systems of the presentinvention can be used in amplification reactions such as real-time PCRreactions. The dual-labeled probes in illustrative examples arehydrolysis probes, such as 5′ nuclease probes (see e.g., Livak et al,PCR Methods Appl., 4:357-562 (1995); and U.S. Pat. No. 5,538,848),molecular beacons (see e.g., Mhlanga, Methods, 25:463-472 (2001)),scorpions (see e.g., Saha, J. Virol. Methods, 93:33-42 (2001)), orhybridizing probes (see e.g., U.S. Pat. No. 7,670,832). In certainembodiments the primers and probes of the present invention are used indigital amplification reactions such as digital PCR reactions.

Primers synthesized by methods of the present invention can be between 5and 50 nucleotides in length and are typically between 10 and 30 andmore typically 15 and 30 nucleotides in length. Probes of the presentinvention can be between 5 and 100, 10 and 50, 10 and 30, or 15 and 30nucleotides in length.

Methods of the present invention can utilize general chemistries andchemical methods known in the art for synthesizing nucleic acidmolecules that include one, two, or more labels, such as a fluorescentlabels. For example, such methods can utilize phosphoramidites and/orsolid supports that are modified to include such labels. Exemplary solidsupports, for example, can include at least one quencher bound through alinker to the solid support. Additional exemplary embodiments canutilize a solid support or a phosphoramidite functionalized moiety thatstabilizes a duplex, triplex or higher order aggregation (e.g.,hybridization) of a nucleic acid molecule synthesized according to thepresent invention with a target nucleic acid molecule.

In certain embodiments, the primers and/or probes of the presentinvention are used in real-time PCR assays such as gene expressionassays or genotyping assays, for example SNP genpotyping assays. Theprobes can be generated using methods provided herein, at aconcentration, for example, of between 1 nM and 1 M, 1 mM and 1 M. Anexemplary concentration can be 100 mM. The probes and/or especially theprimers generated by methods provided herein can be lyophilized. Forexample, 1-1,000,000 picomole of primer can be lyophilized in a reactionvessel, such as a tube, or a well, or can be dried on a spot of an arrayof positions.

In one embodiment, the present invention provides a method for nucleicacid synthesis that includes combining nucleic acid synthesis reactantsinside a microwell and generating the nucleic acid molecule inside themicrowell. The microwell can be linked to a controller, such as acomputer processor, wherein a nucleotide sequence for one or morenucleic acid molecules is input into the controller or otherwise presentin a computer memory of the controller. The controller can be connectedto or otherwise in communication with a nucleic acid molecule design andordering functionality that can be provided over a wide-area network.For example, nucleic acid molecule design and ordering functionality canbe provided over the Internet.

In certain embodiments, methods of the present invention include anHPLC-purification step. In addition, methods of the present inventioncan be performed under ISO and/or GMP-certified conditions. In someembodiment, nucleic acid molecule synthesis is performed using amicrowell plate.

Methods and apparatus of the invention may also be used for thepreparation of libraries. These libraries may contain one or more pointmutations or highly divergent molecules (e.g., nucleic acid moleculeswhich encode proteins with different functional activities). Along theselines, the invention includes methods for the generation of librarieswhere all or some of the library members are chemical synthesized andthus not generated from cellular nucleic acid. Library types which maybe generated by methods of the invention include cDNA libraries, genomiclibraries, combinatorial libraries, point mutation libraries, andcombinations of one or more of such libraries.

As noted above, in some embodiments, the invention includes methods orproducing cDNA library equivalents generated, as well as the librariesthemselves, using bioinformatic information. Using the schematic shownin FIG. 11 for purposes of illustration, a library may be synthesizedand, if necessary, assembled according to methods described herein. Thelibrary members may then be inserted into a non-library nucleic acidmolecule (e.g., a vector, a cellular chromosome, etc.). Insertion may befacilitated by any number of means such as ligation (e.g., “sticky end”ligation).

The invention includes methods for generating library, as well as thelibraries themselves. Some of these libraries are of types which aredifficult or impossible to produce by standard library productionmethods. One such type is a partial cDNA library. Partial cDNA libraries(also referred to as “cDNA equivalent” libraries) may be generated bybioinformatically selecting specific cDNAs for inclusion in the library.Nucleic acid molecules may then be synthesized and, if necessary,assembled to form the library.

cDNA libraries typically contain DNA molecules which correspond to RNAtranscripts within a cell. In many cases, such libraries are biasedtowards transcripts which contain polyA tails. mRNAs represented in suchlibraries typically contain multiple cDNAs corresponding to individualcoding regions. This is true when splice variants of a genomics codingregion are generated by splicing events. The present invention allowsfor the production of cDNA libraries (as well as genomic libraries) with“exclusive” representation. For example, since nucleic acid moleculesare selected for inclusion, as compared to exclusion, the DNA moleculescorresponding to the following may be excluded from libraries: ribosomalRNAs, globin RNAs, tRNAs, and specified mRNAs. Thus, the inventionincludes methods for producing member biased and exclusive memberinclusion cDNA and genomic libraries, as well as the librariesthemselves.

Further, libraries of the invention include those which containspecified nucleic acid molecules. For example, the invention includesmethods for producing cDNA libraries containing a subset of memberrepresented in cDNA libraries generated by standard methods. Forpurposes of illustration, assume that a particular mammalian cell typehas on average 15,000 different mRNA transcripts including splicevariants and one seeks to use a cDNA library which contains 125 cDNAmolecules corresponding to all of the known splice variants oftranscripts corresponding to 35 different kinases. In another instance,one seeks to screen a collection of nucleic acid molecules that encodevariants of the same wild-type coding sequence. Using FIG. 12A forpurposes of illustration, amino acids 85 through 95, and the codingsequence of a wild-type cDNA is shown at the top of the figure. Aminoacids 88 through 91 represent a region which is predicted to be aflexible linker connecting two functional domains. In this instance, acollection of nucleic acid molecules is produced encoding proteins withdifferent, but specified, amino acids at positions 88 through 91 (thelinker region). Collections of nucleic acid molecules such as thoseshown in FIG. 12A may be generated in number of ways.

One way will generally be over inclusive in that additional nucleic acidmolecules will normally be generated. This method employs “dirty bottle”synthesis. To generate variant molecules such as those shown in FIG. 12Areagents for the addition of bases at particular positions are mixed.Thus, when the base at the first and second positions of codon 88 are tobe added, a mixture of reagents for addition of a C and G could be used.The ratio of these reagents may be adjusted to favor either C or Gaddition or the ratio may be adjusted so that equal amounts of C and Gare introduced. In a portion of the population, the codon CGT (arginine)would also be generated.

Another method by which collections of nucleic acid molecules such asthose shown in FIG. 12A may be generated is by synthesizing theindividual variant sequences as separate nucleic acid segments. Thisallows for the generation of only nucleic acid molecules (except forsynthesis errors) which encode the desired variant population members.

The invention also includes individual and collections of nucleic acidmolecules with codon alterations as compared to wild-type molecules, aswell as methods for producing such molecules. In some aspects, a codonaltered library is generated where some or all (in many cases all ormost) of the nucleic acid molecules in the collection are codon alteredas compared to naturally wild-type coding sequences. This shows onesubstantial advantage of methods of the invention over standard libraryconstruction methods. With standard library construction methods,libraries are built from naturally occurring nucleic acid molecules(e.g., genomic DNA, mRNA, etc.). Methods of the invention allow forefficient construction of libraries using bioinformatic information. Theresult being that individual nucleic acid molecules in any collectiongenerated can be generated with “tailored” nucleotide sequences.

Using FIG. 12B for purposes of illustration, a collection of nucleicacid molecules that contain different codons for the same codingsequence may be generated and then screened for desired features (e.g.,increased or decreased expressions levels). Decreased expression levelsmay be desired when over expression of a protein is delirious to cellsor host organisms that the protein is produced in. Thus, codon selectioncan be used as an expression regulation mechanism.

Methods of the invention may also be used to generate large numbers ofprimers for multiplex amplification (e.g., PCR). Typically such primerswill be between 15 and 100 (e.g., from 15 to 90, from 25 to 90, from 25to 80, from 25 to 70, from 25 to 60, from 25 to 50, from 30 to 90, from30 to 60, etc.) nucleotides in length. Further, primers may also containbar codes to allow for the tagging of amplified nucleic acid moleculesfor, for example, later identification as well as tracking of primersand primer pairs during and subsequent to synthesis runs.

In some instances, between 500 and 50,000, between 1,000 and 50,000,between 2,000 and 50,000, between 5,000 and 50,000, between 5,000 and40,000, between 5,000 and 30,000, between 5,000 and 100,000, between5,000 and 300,000, between 5,000 and 500,000, between 5,000 and1,000,000, between 5,000 and 5,000,000, between 10,000 and 100,000,between 10,000 and 500,000, between 10,000 and 800,000, between 20,000and 100,000, between 20,000 and 500,000, etc. primers pairs will begenerated.

The invention includes the preparation of primers which may be used inprocesses such as Life Technology Corporation's AMPLISEQ™ products (see,e.g., cat. no. 4472395). Products such as this employ multiplex PCR forthe amplification of specific nucleic acid molecules. The amplifiednucleic acid molecules may then be used in downstream processes such assequencing to identify nucleic acids present in a starting sample. Insome cases, modified nucleic acid bases and/or natural bases nottypically associated with DNA (e.g., deoxyuridine) are syntheticallyincorporated into the primer sequences as a “fifth (or greater) bottle”to impart particular properties into the individual primer(s) and/orprimer set to facilitate downstream processing of the amplified productsprior to sequencing or to further impart encoding of the individualprimer(s) and/or primer set in the manner of barcoding to facilitate andresolve complex sequence analysis typically from a mixture of samples.

The invention thus provides methods for producing primer pools, as wellas the primer pools themselves. Primer pools may be used to amplify RNAand/or DNA populations or subpopulation. As an example, primer pools maybe produced that allow for the amplification of genomic DNA representingthe entire nuclear genome of a cell, a single nuclear chromosome, a setof nuclear genes or regions (e.g., a set of chromosomal loci), amitochondrial genome, or a chloroplast genome, as well as combinationsthereof. The invention thus includes the bioinformatic design of primersfor specific applications (e.g., the applications set out immediatelyabove).

The invention also provides methods for producing primer pools for theamplification of specific RNA populations. In one embodiment of theinvention, a primer pool is designed to amplify all mRNA molecules or asubpopulation of mRNA molecules (e.g., mRNAs encoding kinases,phosphatases, etc.) produced by a cell but, optionally, not other RNAmolecules (e.g., tRNA, rRNA, hnRNA, etc.). Such primer pools may then beused for expression analysis (e.g., measuring the level of expressionunder various conditions). Expression analysis may be performed using,for example, microarrays or sequencing platforms. The invention thusincludes expression analysis methods. In some embodiments, such methodsinclude one or more of the following steps: (a) designingbioinformatically a primer pool, (b) synthesizing primer pairs of theprimer pool, (c) contacting the primer pool to a sample derived from acell containing nucleic acids (e.g., mRNA), (d) amplifying nucleic acidmolecules in the sample corresponding to the primer pairs, and (e)analyzing the resulting amplified nucleic acid molecules.

The reduction or elimination of nucleic acid molecules corresponding torDNA is desirable in many expression analysis applications because ofthe abundance of rRNA in many samples. Other rRNA amplificationreduction methods are set out in U.S. Patent Publication No.2008/0187969, the disclosure of which is incorporated herein byreference.

The invention also includes variations of the above for additionalapplications such as multiplex methods of the identification ofmutations in genomic nucleic acid. Thus, the invention further includesmethods and compositions for the identification of mutations, includingcancer screens.

The invention includes methods for producing various numbers of primer(in many instances in primer pairs). The number of primers which may beprepared by methods of the invention as separate entities and/or inmixed populations range from five to 500,000, from 500 to 500,000, from1,000 to 500,000, from 5,000 to 500,000, from 10,000 to 500,000, from20,000 to 500,000, from 30,000 to 500,000, from 5,000 to 250,000, from5,000 to 100,000, from five to 5,000, from five to 50,000, from 5,000 to800,000, from 5,000 to 1,000,000, from 5,000 to 2,000,000, from 10,000to 2,000,000, from 20,000 to 1,000,000, from 30,000 to 2,000,000, etc.

The invention thus provides methods for the rapid design, configurationand synthesis of defined sets of primers for the specificallydetermining genetic compositions and characterization of regions for awide variety of analyses, sample sets and experimental designs. Thisaspect of the invention partially flows from the use of bioinformaticsin conjunction with nucleic acid molecule synthesis methods describedherein. In particular, the complete sequences of a considerable numberof genomes have been sequenced. This sequence information, combined withnucleic acid synthesis methods (as well as other methods) describedherein allow for detailed genome and transcriptome analyses. Multiplexmethods, such as those set out above, provide one means for performingsuch analyses.

Representative Embodiments

Numerous variations of the invention are feasible and may be employed toachieve the desired results. Many such variations may be directed todesign features. In some instances, such design features may be used foroperator convenience and/or cost savings (e.g., decreased reagentusage).

FIG. 9 shows one embodiment of an electrical coil that may be used inspecific embodiments of the invention. Numerous variations of suchcoils, a number of which are described elsewhere herein, may be usedwith the invention.

An electrical coil such as that shown in FIG. 9 may be designed with thefollowing exemplary structural an operation parameters: Maximum currentdensity 3 Amps/mm², double layer flat coil, wire cross section 5×2 μm,10 turns, inner diameter (Di)˜10 μm, outer diameter (Da)˜180 μm, andwire length ˜6 mm.

TABLE 6 current Mag. Field Strength (A/m) (A) (approx. short coil)0.00003 6.314390318 current (μA) Mag. Flux Density (T) 30 7.9349E−06

FIG. 9 and Table 6 show exemplary specifications of a flat double layercoil that can be build up on a wafer. A coil such as that shown in FIG.9 may be designed such that contact is made with each well in asynthesis platform. Further, the generation of a magnetic field may beused to lift beads from synthesis sites (e.g., wells). Exemplarymagnetic field strength/flux density figures are shown in Table 6. AFEM-program like Comsol (www.comsol.com) may be used to calculateparameters for specific systems and formats.

Several materials, and properties associated with these materials, thatmay be used in electrodes used in various aspects of the invention areset out in Table 7. The selection of electrode materials will bedetermined by numerous factors including costs and various designspecifications and power requirements.

TABLE 7 Coil DC Specific Resistance Resistance Power Voltage Material((Ω*mm²)/m) (Ω) (μW) (V) Copper 1.68E−02 10.068 0.009061 0.000302Aluminum 2.65E−02 15.9 0.01431 0.000477 Gold 2.21E−02 13.284 0.0119560.000399

Electrodes (e.g., electrical coils) used in the practice of theinvention will be designed so as to meet the particular applications forwhich they are used. As an example, when electrodes are used to generateEGA, they will generally be designed with the following in mind: (1) Theapplication (e.g., local application) of sufficient current to allow forthe generation of an effective amount of EGA within a specified timeperiod, (2) limitation of heating associated with the application ofcurrent. Thus, will generally be desirable to limit the amount ofcurrent used to reach a local pH of 1.0 with the addition of littleexcess current. Table 8 provides calculations for achieving this withspecific well parameters. Further, the generation of pH 1 in a well asset out below will require that 727 pA of current be applied for about 1second. This results in a current density of 115 A/m² on the workingelectrode.

TABLE 8 Current/pH generation for a cylindrical well using a 2.8 μm beadInput Parameter Well Diameter (μm) 4 Well Height (μm) 3 Desired pH 1Buffer Concen. (mol/L) 0.1 Area (μm²) 6.3 Well Vol. (μm³) 37.68 ProtonsGenerated 4.52 × 10⁹ Charge (pAs) 727 Current Density WE (A/m²) 115

The shape of an electrode may vary greatly and may be a coil as shown inFIG. 9, a disk, a thin film, etc. Further, electrodes used in thepractice of the invention may be composed of any number of compounds,including platinum, palladium, copper, gold, aluminum, niobium, niobiumoxide, tungsten, titanium, tantalum, molybdenum, nickel, platinum,silver, manganese, neodymium, carbon, and silicon, and an alloy materialor a compound material containing one or more of the above-describedelements, as well as other elements.

FIG. 10 shows an exemplary apparatus format of the invention. Thisfigure shows two pumps 1000 that deliver fluids, as well as gases whenappropriate, through tubes 1001 to fluidic channels 1002, which isbounded at the top by a plate 1007. Fluids delivered to the apparatusare removed through drainage channels 1003 to drainage tubes 1004 whichlead to waste collection 1005. The pumps 1000 are connected to fluidreservoirs (not shown), or gas reservoirs when appropriate, and acontrol device (not shown) that regulate what fluid or gas is deliveredto the apparatus.

The control device also regulates the length of time that fluids orgasses contact nucleic acid synthesis “chips” 1006. Three nucleic acidsynthesis “chips” 1006 are visible in FIG. 10 resting on an electrode1008. Fluids and/or gases are put in contact with the chips and currentpasses through particular locations on the chips where it is desirablefor chemical reactions to occur. As described elsewhere herein, anynumber of reagents and washing materials may be used in the practice ofthe invention. In many instances, the reagents and materials used willbe those which allow for the production of nucleic acid molecules.

The lower electrode 1008, as shown in FIG. 9, covers the entire base ofthe apparatus. This need not be the case and one or more electrodes maybe associated with one end of each well or more than one well. Oppositethis electrode (shown as a lower electrode 1008 in FIG. 10), there willtypically be one or more second electrodes (not shown in FIG. 10) thatallows for current to flow through entire chips or through wells of thechips. In many instances, these second electrodes will be positionedover individual wells of the chip to allow for current to be directedthrough the wells on an individual basis (see FIGS. 2A-2B).

Fluid channel 1002 can be formed in a surface layer. The surface layercan be formed of a polymeric material, inorganic material, or acombination thereof. For example, the surface layer can be formed of apolymeric material. An exemplary polymeric material includes acrylic,fluoropolymer, ethylene vinyl acetate (EVA), or any combination thereof.In an example, the polymeric material is a fluoropolymer. An exemplaryfluoropolymer includes polyvinylidene fluoride (PVDF), polyvinylfluoride (PVF), fluorinated ethylene propylene (FEP) copolymer, ethylenechlorotrifluoroethylene (ECTFE) copolymer, a copolymer oftetrafluoroethylene, hexafluoropropylene, and vinylidene fluoride (THV),a copolymer of tetrafluoroethylene and perfluoro methylvinylether (PFAor MFA), a fluoropolymer having a fluorinated oxolane in its backbone,perfluoroether, or any combination thereof. In particular, thefluoropolymer can be a fluoropolymer having fluorinated oxolane in itsbackbone, for example, Cytop. Further, the polymer coating can beamorphous, exhibiting little or no crystallinity. In another example,the surface layer is formed of an inorganic insulator. For example, theinorganic insulator can include an oxide of silicon, aluminum, hafnium,tantalum, zirconium, or any combination thereof, can includetetraorthosilicate, can include a nitride of silicon, or can include anycombination thereof. In an example, the inorganic insulator can includean oxide of silicon. In another example, the inorganic insulatorincludes a nitride of silicon.

The surface layer can have a thickness in a range of 0.3 micrometers to10 micrometers, such as a range of 0.5 micrometers to 6 micrometers.

Individual wells used in the practice of the invention may be of anynumber of shapes and sizes. One example of well parameters is set out inTable 9. Of course, well volume and other factors will change with welldimensions.

TABLE 9 Exemplary cylindrical well parameters Input Parameter WellDiameter 40 μm Well Height 35 μm pH 1 Buffer Concen. (mol/l) 0.1 μmArea/Well 1256 μm² Output Parameter Well Volume 43,960 μm³ GeneratedProtons/Well (incl. buffer) 5.28 × 10¹² Charge 848 nAs CurrentDensity/Well 675 A/m²

TABLE 10 Input Output Number of Oligos/ 1.0 × 10⁸ Required Buffer1,666,667 Bead Vol. (pl) Number of beads with 1 same oligo sequenceOligonucleotide 0.10 Concentration (μmol/l) Bead Diameter (μm) 30 BeadVol. (μm³) 14,137.17

Table 10 shows some bead parameters and estimate buffer volume andconcentration for a particular bead size.

After completion of nucleic acid molecules production steps, thesubstrates (e.g., beads) containing the nucleic acid molecules may becollected, separated from the synthesis substrates, and furtherprocessed.

An exemplary work flow is one such as the following: (1) Beads areprepared with functional (hydroxyl or amine) groups, (2) the beads arederivatized in batch off-line forming amide with pre-synthesizeduniversal primers with rare type IIs restriction site for enzymaticcleavage of synthesized nucleic acid molecules off the beads, (3) thebeads are loaded by flowing suspension into chip, application of currentsecures beads in wells, (4) the loaded beads are in or near physicalcontact with an anode and EGA is generated at anode and on the beadsurface for deprotection, (5) synthesis steps as described herein areperformed, (6) after synthesis, digitally electro-eject of desired beadsfrom well is accomplished by reversing the current, (7) ejected beadsare collected and pooled from the liquid flow out of chip, and (8) otherbeads are held in wells until later in time by the application of weakcurrent in initial anode/cathode orientation.

Those skilled in the art will recognize that the operations of thevarious embodiments may be implemented using hardware, software,firmware, or combinations thereof, as appropriate. For example, someprocesses can be carried out using processors or other digital circuitryunder the control of software, firmware, or hard-wired logic. (The term“logic” herein refers to fixed hardware, programmable logic and/or anappropriate combination thereof, as would be recognized by one skilledin the art to carry out the recited functions.) Software and firmwarecan be stored on computer-readable media. Some other processes can beimplemented using analog circuitry, as is well known to one of ordinaryskill in the art. Additionally, memory or other storage, as well ascommunication components, may be employed in embodiments of theinvention.

FIG. 15 is a block diagram that illustrates a computer system 1500 thatmay be employed to carry out processing functionality, according tovarious embodiments, upon which embodiments of a thermal cycler system500 of FIG. 5 may utilize. Computing system 1500 can include one or moreprocessors or controllers, such as a processor 1504. Processor 1504 canbe implemented using a general or special purpose processing engine suchas, for example, a microprocessor, controller or other control logic. Inthis example, processor 1504 is connected to a bus 1502 or othercommunication medium. For example, processor 1504 may be a currentcontroller as described above with reference to FIGS. 2A-2B.

Further, it should be appreciated that a computing system 1500 of FIG.12 may be embodied in any of a number of forms, such as a rack-mountedcomputer, mainframe, supercomputer, server, client, a desktop computer,a laptop computer, a tablet computer, hand-held computing device (e.g.,PDA, cell phone, smart phone, palmtop, etc.), cluster grid, netbook,embedded systems, or any other type of special or general purposecomputing device as may be desirable or appropriate for a givenapplication or environment. Additionally, a computing system 1500 caninclude a conventional network system including a client/serverenvironment and one or more database servers, or integration withLIS/LIMS infrastructure. A number of conventional network systems,including a local area network (LAN) or a wide area network (WAN), andincluding wireless and/or wired components, are known in the art.Additionally, client/server environments, database servers, and networksare well documented in the art.

Computing system 1500 may include bus 1502 or other communicationmechanism for communicating information, and processor 1504 coupled withbus 1502 for processing information.

Computing system 1500 also includes a memory 1506, which can be a randomaccess memory (RAM) or other dynamic memory, coupled to bus 1502 forstoring instructions to be executed by processor 1504. Memory 1506 alsomay be used for storing temporary variables or other intermediateinformation during execution of instructions to be executed by processor1504. Computing system 1500 further includes a read only memory (ROM)1508 or other static storage device coupled to bus 1502 for storingstatic information and instructions for processor 1504.

Computing system 1500 may also include a non-transitory storage device1510, such as a magnetic disk, optical disk, or solid state drive (SSD)is provided and coupled to bus 1502 for storing information andinstructions. Storage device 1510 may include a media drive and aremovable storage interface. A media drive may include a drive or othermechanism to support fixed or removable storage media, such as a harddisk drive, a floppy disk drive, a magnetic tape drive, an optical diskdrive, a CD or DVD drive (R or RW), flash drive, or other removable orfixed media drive. As these examples illustrate, the storage media mayinclude a computer-readable storage medium having stored there inparticular computer software, instructions, or data.

In alternative embodiments, storage device 1510 may include othersimilar instrumentalities for allowing computer programs or otherinstructions or data to be loaded into computing system 1500. Suchinstrumentalities may include, for example, a removable storage unit andan interface, such as a program cartridge and cartridge interface, aremovable memory (for example, a flash memory or other removable memorymodule) and memory slot, and other removable storage units andinterfaces that allow software and data to be transferred from thestorage device 1510 to computing system 1500.

Computing system 1500 can also include a communications interface 1518.Communications interface 1518 can be used to allow software and data tobe transferred between computing system 1500 and external devices.Examples of communications interface 1518 can include a modem, a networkinterface (such as an Ethernet or other NIC card), a communications port(such as for example, a USB port, a RS-232C serial port), a PCMCIA slotand card, Bluetooth, etc. Software and data transferred viacommunications interface 1518 are in the form of signals which can beelectronic, electromagnetic, optical or other signals capable of beingreceived by communications interface 1518. These signals may betransmitted and received by communications interface 1518 via a channelsuch as a wireless medium, wire or cable, fiber optics, or othercommunications medium. Some examples of a channel include a phone line,a cellular phone link, an RF link, a network interface, a local or widearea network, and other communications channels.

Computing system 1500 may be coupled via bus 1502 to a display 1512,such as a cathode ray tube (CRT) or liquid crystal display (LCD), fordisplaying information to a computer user. An input device 1514,including alphanumeric and other keys, is coupled to bus 1502 forcommunicating information and command selections to processor 1504, forexample. An input device may also be a display, such as an LCD display,configured with touch screen input capabilities. Another type of userinput device is cursor control 1516, such as a mouse, a trackball orcursor direction keys for communicating direction information andcommand selections to processor 1504 and for controlling cursor movementon display 1512. This input device typically has two degrees of freedomin two axes, a first axis (e.g., x) and a second axis (e.g., y), thatallows the device to specify positions in a plane. A computing system1500 provides data processing and provides a level of confidence forsuch data. Consistent with certain implementations of embodiments of thepresent teachings, data processing and confidence values are provided bycomputing system 1500 in response to processor 1504 executing one ormore sequences of one or more instructions contained in memory 1506.Such instructions may be read into memory 1506 from anothercomputer-readable medium, such as storage device 1510. Execution of thesequences of instructions contained in memory 1506 causes processor 1504to perform the process states described herein. Alternatively hard-wiredcircuitry may be used in place of or in combination with softwareinstructions to implement embodiments of the present teachings. Thusimplementations of embodiments of the present teachings are not limitedto any specific combination of hardware circuitry and software.

The term “computer-readable medium” and “computer program product” asused herein generally refers to any media that is involved in providingone or more sequences or one or more instructions to processor 1504 forexecution. Such instructions, generally referred to as “computer programcode” (which may be grouped in the form of computer programs or othergroupings), when executed, enable the computing system 1500 to performfeatures or functions of embodiments of the present invention. These andother forms of computer-readable media may take many forms, includingbut not limited to, non-volatile media, volatile media, and transmissionmedia. Non-volatile media includes, for example, solid state, optical ormagnetic disks, such as storage device 1510. Volatile media includesdynamic memory, such as memory 1506. Transmission media includes coaxialcables, copper wire, and fiber optics, including the wires that comprisebus 1502.

Common forms of computer-readable media include, for example, a floppydisk, a flexible disk, hard disk, magnetic tape, or any other magneticmedium, a CD-ROM, any other optical medium, punch cards, paper tape, anyother physical medium with patterns of holes, a RAM, PROM, and EPROM, aFLASH-EPROM, any other memory chip or cartridge, a carrier wave asdescribed hereinafter, or any other medium from which a computer canread.

Various forms of computer readable media may be involved in carrying oneor more sequences of one or more instructions to processor 1504 forexecution. For example, the instructions may initially be carried onmagnetic disk of a remote computer. The remote computer can load theinstructions into its dynamic memory and send the instructions over atelephone line using a modem. A modem local to computing system 1500 canreceive the data on the telephone line and use an infra-red transmitterto convert the data to an infra-red signal. An infra-red detectorcoupled to bus 1502 can receive the data carried in the infra-red signaland place the data on bus 1502. Bus 1502 carries the data to memory1506, from which processor 1504 retrieves and executes the instructions.The instructions received by memory 1506 may optionally be stored onstorage device 1510 either before or after execution by processor 1504.

It will be appreciated that, for clarity purposes, the above descriptionhas described embodiments of the invention with reference to differentfunctional units and processors. However, it will be apparent that anysuitable distribution of functionality between different functionalunits, processors or domains may be used without detracting from theinvention. For example, functionality illustrated to be performed byseparate processors or controllers may be performed by the sameprocessor or controller. Hence, references to specific functional unitsare only to be seen as references to suitable means for providing thedescribed functionality, rather than indicative of a strict logical orphysical structure or organization.

Embodiments may be in accordance with following numbered clauses:

1. A multiwell plate for non-template directed synthesis of nucleic acidmolecules, the plate comprising:

(a) at least one bead located in each of a plurality of wells of theplate, and

(b) an electrochemically generated acid being present in one or morewell,

wherein the bead is between 0.1 μm and 100 μm in diameter.

2. The multiwell plate of clause 1, wherein the number of wells in theplate is between 10 and 10,000,000.

3. The multiwell plate of any one of the preceding clauses, wherein thetotal volume of each well is between 0.1 μl and 50 μl.

4. The multiwell plate of any one of the preceding clauses, wherein eachwell is operably connected to a pair of electrodes.

5. The multiwell plate of any one of the preceding clauses, wherein thewells of the plate are connected to microfluidic channels for theintroduction and removal of reagents.

6. A method for the generation of an assembled nucleic acid molecule,the method comprising:

-   -   (a) synthesizing a plurality of nucleic acid molecules, wherein        each nucleic acid molecule is prepared in a well of a plate in        an average amount of from about 0.001 nanomoles to about 1,000        nanomoles;    -   (b) combining the nucleic acid molecules generated in (a) to        produce a pool;    -   (c) joining some or all of the nucleic acid molecules present in        the pool formed in (b) to form a plurality of larger nucleic        acid molecules;    -   (d) eliminating nucleic acid molecules which contain sequence        errors from the plurality of larger nucleic acid molecules        formed in (c) to produce an error corrected nucleic acid        molecule pool; and    -   (e) assembling the nucleic acid molecules in the error corrected        nucleic acid molecule pool to form the assembled nucleic acid        molecule.

7. The method of clause 6, wherein the joining in (c) is mediated bypolymerase chain reaction and/or ligases.

8. The method of any one of clauses 6 or 7, wherein the assemblednucleic acid molecule is composed of at least five nucleic acidmolecules.

9. The method of any one of clauses 6 to 8, wherein the assemblednucleic acid molecule is composed of between five and five thousandnucleic acid molecules.

10. The method of any one of clauses 6 to 9, wherein the assemblednucleic acid molecule is at least 20 kilobases.

11. The method of any one of clauses 6 to 10, wherein the assemblednucleic acid molecule is between 10 kilobases and 1 megabase.

12. The method of any one of clauses 6 to 11, wherein the assemblednucleic acid molecule is closed, circular.

13. The method of any one of clauses 6 to 12, wherein the assemblednucleic acid molecule is a plasmid.

14. The method of any one of clauses 6 to 13, wherein two or moreassembled nucleic acid molecule are simultaneously formed.

15. The method of any one of clauses 6 to 14, wherein assembly of thenucleic acid molecules in the error corrected nucleic acid molecule pooloccurs in a fungal cell.

16. The method of any one of clauses 6 to 15, wherein step (b) furthercomprises combining nucleic acid molecules generated in (a) with nucleicacid molecules obtained by other means to form a pool, wherein saidother means include PCR, restriction enzyme digest or exonucleasetreatment.

17. The method of any one of clauses 6 to 16, wherein the assemblednucleic acid molecule generated in (e) is assembled and introduced intoa cloning vector.

18. A method for producing a product nucleic acid molecule, the methodcomprising:

(a) designing the product nucleic acid molecule of between 0.1 kilobasesand 500 kilobases in size, wherein the product nucleic acid molecule isdefined by nucleotide sequence;

(b) synthesizing a plurality of individual nucleic acid molecules whichdiffer in nucleotide sequence, wherein each individual nucleic acidmolecule is synthesized to prepare a quantity of between 1.0×10³ and1.0×10⁹ copies and wherein the individual nucleic acid molecules arecapable of hybridizing with one or more of the other individual nucleicacid molecules;

(c) combining the individual nucleic acid molecules synthesized in (b)under conditions which allow for hybridization of the individual nucleicacid molecules under conditions which allow for the formation of atleast one larger nucleic acid molecule; and

(d) combining the at least one larger nucleic acid molecule formed in(c) with one or more additional nucleic acid molecules to form theproduct nucleic acid molecule, wherein the product nucleic acid moleculecontains less than one sequence error per kilobase.

19. The method of clause 18, wherein the product nucleic acid moleculeis of a size selected from the groups consisting of:

(a) between 0.1 kilobases and 300 kilobases;

(b) between 10 kilobases and 200 kilobases;

(c) between 10 kilobases and 100 kilobases; and

(d) between 10 kilobases and 50 kilobases.

20. The method of any one of clauses 18 or 19, wherein an errorcorrection process is employed after step (b) or after step (d).

21. The method of any one of clauses 18 to 20, wherein each individualnucleic acid molecule is synthesized to prepare a quantity selected fromthe group consisting of:

(a) between 5.0×10³ and 1.0×10⁹ copies;

(b) between 1.0×10⁶ and 1.0×10⁹ copies;

(c) between 1.0×10⁷ and 1.0×10⁸ copies;

(d) between 2.0×10⁷ and 1.0×10⁹ copies;

(e) between 5.0×10⁷ and 1.0×10⁹ copies;

(f) between 7.0×10⁷ and 1.0×10⁹ copies;

(g) between 2.0×10⁷ and 8.0×10⁸ copies; and

(h) between 2.0×10⁷ and 5.0×10⁸ copies.

22. The method of any one of clauses 18 to 21, wherein polymerase chainreactions are used to amplify the at least one larger nucleic acidmolecule formed in step (c).

23. The method of any one of clauses 18 to 22, wherein the productnucleic acid molecule is self replicable.

24. The method of any one of clauses 18 to 23, wherein the selfreplicable nucleic acid molecule is a plasmid.

25. The method of any one of clauses 18 to 24, wherein the individualnucleic acid molecules are synthesized on beads, wherein each bead iscontaining in a well.

26. The method of any one of clauses 18 to 25, wherein the beads are ofa size selected from the group consisting of:

(a) between 5 μm and 100 μm in diameter;

(b) between 20 μm and 100 μm in diameter;

(c) between 28 and 32 μm in diameter;

(d) between 5 μm and 60 μm in diameter; and

(e) between 10 μm and 100 μm in diameter.

27. A method for the generation of a self replicating nucleic acidmolecule, the method comprising:

(a) synthesizing a plurality of nucleic acid molecules, wherein eachnucleic acid molecule is prepared in a microquantity in the well of aplate;

(b) joining some or all of the nucleic acid molecules present in thepool formed in (a) to form a plurality of larger nucleic acid molecules;and

(c) assembling the plurality of larger nucleic acid molecules to formthe self replicating nucleic acid molecule.

28. The method of clause 27, wherein the self replicating nucleic acidmolecule is a chromosome or a plasmid.

29. The method of any one of clauses 27 or 28, wherein the selfreplicating nucleic acid molecule is a genome.

30. The method of any one of clauses 27 to 29, wherein the genome is aviral genome, a nuclear genome, an organelle genome, or a genome of aprokaryotic cell.

31. A method for synthesizing and assembling a nucleic acid moleculewhich encodes more than one expression product, the method comprising:

(a) synthesizing a plurality of nucleic acid molecules, wherein eachnucleic acid molecule is prepared in a microquantity;

(b) joining some or all of the nucleic acid molecules present in thepool formed in (a) to form a plurality of larger nucleic acid molecules;and

(c) assembling the plurality of larger nucleic acid molecules to formthe nucleic acid molecule which encodes more than one expressionproduct.

32. The method of clause 31, wherein the more than one expressionproducts are proteins that are involved in the same biological pathway.

33. The method of any one of clauses 31 or 32, wherein the more than oneexpression products are proteins which are involved in the samebiological pathway are enzymes that catalyze a series of chemicalreactions in a biological pathway.

34. The method of any one of clauses 31 to 33, wherein the chemicalreactions in the same biological pathway are sequential reactions.

35. The method of any one of clauses 31 to 34, wherein the biologicalpathway results in an end product selected from the group consisting of:

(a) a biofuel precursor;

(b) an antibiotic or antibiotic precursor;

(c) a food component; and

(d) an industrial enzyme.

36. The method of any one of clauses 31 to 35, wherein the biofuelprecursor is an alcohol selected from the group consisting of:

(a) butanol;

(b) pentanol;

(c) hexanol;

(d) heptanol; and

(e) octanol.

37. The method of any one of clauses 31 to 36, wherein the foodcomponent is an amino acid selected from the group consisting of:

(a) L-lysine;

(b) L-threonine;

(c) L-methionine;

(d) L-leucine;

(e) L-isoleucine:

(f) L-valine, and

(g) Homoserine.

38. The method of any one of clauses 31 to 37, wherein the assemblednucleic acid molecule is introduced into a prokaryotic cell.

39. The method of any one of clauses 31 to 38, wherein the prokaryoticcell is a Corynebacterium.

40. The method of any one of clauses 31 to 39, wherein theCorynebacterium is Corynebacterium glutamicum.

41. A non-transitory computer-readable storage medium encoded withinstructions, executable by a processor, for generating assemblednucleic acid molecule, the instructions comprising instructions for:

(a) synthesizing a plurality of nucleic acid molecules, wherein eachnucleic acid molecule is prepared in a microquantity in the well of aplate;

(b) combining the nucleic acid molecules generated in (a) to produce apool;

(c) joining some or all of the nucleic acid molecules present in thepool formed in (b) to form a plurality of larger nucleic acid molecules;

(d) eliminating nucleic acid molecules which contain sequence errorsfrom the plurality of larger nucleic acid molecules formed in (c) toproduce an error corrected nucleic acid molecule pool; and

(e) assembling the nucleic acid molecules in the error corrected nucleicacid molecule pool to form the assembled nucleic acid molecule.

42. A system for generating assembled nucleic acid molecule, the systemcomprising:

a processor; and

a memory encoded with processor-executable instructions for:

-   -   (a) synthesizing a plurality of nucleic acid molecules, wherein        each nucleic acid molecule is prepared in a microquantity in the        well of a plate;    -   (b) combining the nucleic acid molecules generated in (a) to        produce a pool;    -   (c) joining some or all of the nucleic acid molecules present in        the pool formed in (b) to form a plurality of larger nucleic        acid molecules;    -   (d) eliminating nucleic acid molecules which contain sequence        errors from the plurality of larger nucleic acid molecules        formed in (c) to produce an error corrected nucleic acid        molecule pool; and    -   (e) assembling the nucleic acid molecules in the error corrected        nucleic acid molecule pool to form the assembled nucleic acid        molecule.

All publications, patents and patent applications mentioned in thisSpecification are indicative of the level of skill of those of ordinaryskill in the art and are herein incorporated by reference to the sameextent as if each individual publication, patent, or patent applicationswas specifically and individually indicated to be incorporated byreference.

The invention being thus described, one skilled in the art wouldrecognize that the invention may be varied in many ways. Such variationsare not to be regarded as a departure from the spirit and scope of theinvention, and all such modifications as would be obvious to one ofordinary skill in the art are intended to be included within the scopeof the following claims.

1. A multiwell plate for non-template directed synthesis of nucleic acidmolecules, the plate comprising: (a) a magnetic bead located in each ofa plurality of wells of the plate, and (b) an electrochemicallygenerated acid being present in one or more well, wherein the bead isbetween 1.0 μm and 100 μm in diameter. 2.-3. (canceled)
 4. The multiwellplate of claim 1, wherein each well is operably connected to a pair ofelectrodes.
 5. (canceled)
 6. A method for the generation of an assemblednucleic acid molecule, the method comprising: (a) synthesizing aplurality of nucleic acid molecules, wherein each nucleic acid moleculeis prepared in a well of a plate in an average amount of from about0.001 nanomoles to about 1,000 nanomoles; (b) combining the nucleic acidmolecules generated in (a) to produce a pool; (c) joining some or all ofthe nucleic acid molecules present in the pool formed in (b) to form aplurality of larger nucleic acid molecules; (d) eliminating nucleic acidmolecules which contain sequence errors from the plurality of largernucleic acid molecules formed in (c) to produce an error correctednucleic acid molecule pool; and (e) assembling the nucleic acidmolecules in the error corrected nucleic acid molecule pool to form theassembled nucleic acid molecule.
 7. The method of claim 6, wherein thejoining in (c) is mediated by polymerase chain reaction and/or ligases.8.-41. (canceled)
 42. A method for producing a plurality of nucleic acidmolecules, the method comprising: (a) synthesizing the plurality ofnucleic acid molecules on separate beads in wells of a plate, whereineach well is configured to accommodate a single bead, wherein the volumeof each well is between 0.1 picoliters and 10 nanoliters, wherein thewells comprise an electrode for the production of electrochemicallygenerated acid, and wherein the nucleic acid molecules synthesized oneach bead are designed to have the same nucleotide sequence; (b)selectively removing a plurality of beads from the wells, wherein thebeads removed from the wells comprise a first set of individual nucleicacid molecules, wherein beads comprising nucleic acid molecules that arenot part of the first set of individual nucleic acid molecules are notremoved from their respective wells; and (c) collecting the beadsselectively removed from wells in step (b) in a vessel.
 43. The methodof claim 42, further comprising: (e) selectively removing a plurality ofbeads from the wells, wherein the beads removed from the wells comprisea second set of individual nucleic acid molecules, wherein beadscomprising nucleic acid molecules that are not part of the second set ofindividual nucleic acid molecules are not removed from their respectivewells; and (f) collecting the beads selectively removed from wells instep (e) in a vessel.
 44. The method of claim 42, wherein the number ofbeads collected in step (c) is between 100 to 1,000.
 45. The method ofclaim 42, further comprising releasing the nucleic acid molecules fromthe beads collected in the vessel in step (c).
 46. The method of claim42, wherein the first set of individual nucleic acid molecules areprimers.
 47. The method of claim 46, wherein the primers are 5 to 100nucleotides in length.
 48. The method of claim 46, wherein the primersare lyophilized.
 49. The method of claim 46, wherein the vessel is awell of a multiwell plate.
 50. The method of claim 42, wherein themembers of the first set of nucleic acid molecules are between 40 to 200nucleotides in length.
 51. The method of claim 42, wherein the beads inthe wells are porous beads.
 52. The method of claim 42, wherein thebeads in the wells of the plate are monodispersed beads.
 53. The methodof claim 52, wherein the monodispersed beads are between 10 μm and 40 μmin diameter.
 54. The method of claim 42, wherein synthesis of thenucleic acid molecules on the separate beads involves deblocking in step(a) by an acid generated by in a redox reaction mixture in the wells ofthe plate.
 55. The method of claim 54, wherein the redox reactionmixture in the wells of the plate contains hydroquinone.
 56. The methodof claim 55, wherein the redox reaction mixture in the wells of theplate contains hydroquinone and anthraquinone.
 57. The method of claim54, wherein the electrochemically generated acid is generated in thewell by passing an electrical current through the wells.